Methods, compositions and kits for treating a subject using a recombinant heteromultimeric neutralizing binding protein

ABSTRACT

Methods, compositions and kits are provided for treating a subject exposed to or at risk for exposure to a disease agent using a pharmaceutical composition including at least one recombinant heteromultimeric neutralizing binding protein including two or multiple binding regions, such that the binding regions are not identical, and each binding region specifically binds a non-overlapping portion of the disease agent, thereby treating the subject for exposure to the disease agent. In a related embodiment, the heteromultimeric neutralizing binding protein includes two or multiple binding regions that neutralize a plurality of disease agents. In certain embodiments, the disease agent includes a bacterium, a bacterial protein, a virus, a viral protein, a cancer cell, and a protein or molecule produced therefrom. In certain embodiments, the disease agent is a plurality of different disease agents.

RELATED APPLICATIONS

This application claims the benefit of U.S. provisional application Ser.No. 61/514,949 filed Aug. 4, 2011 entitled, “Methods, compositions andkits for treating a subject using a recombinant heteromultimericneutralizing binding protein”, inventors Charles B. Shoemaker andHanping Feng, and is a continuation-in-part of U.S. utility applicationSer. No. 12/889,511 filed Sep. 24, 2010, which is a continuation-in-partapplication of U.S. utility application Ser. No. 12/032,744 filed Feb.18, 2008, which claims the benefit of U.S. provisional application Ser.No. 60/890,626 filed Feb. 20, 2007, each of which is incorporated byreference herein in its entirety.

GOVERNMENT SUPPORT

This invention was made with government support under grant U54-AI057159awarded by the National Institutes of Health. The government has certainrights in the invention.

TECHNICAL FIELD

Compositions, methods, and kits using a recombinant heteromultimericneutralizing binding protein are provided for treating a subject at riskfor exposure or exposed to a disease agent.

BACKGROUND

A need exists for generating high affinity binding agents that treatboth routine incidents of disease and pandemics, and efforts to discoverand produce these agents are underway. The production of antibodies andtheir storage is a costly and lengthy process. In fact, development of asingle antibody therapeutic agent often requires years of clinicalstudy. Yet multiple, different therapeutic antibodies are necessary forthe effective treatment of patients exposed to a disease agent, aninfection outbreak or a bio-terrorist assault. Developing and producingmultiple antibodies that can bind to different targets (e.g. microbialpathogens, viral pathogens, toxins, and cancer cells) is often adifficult task because it involves separately producing, storing andtransporting multiple antibodies for each pathogen or toxin. Productionand stockpiling a sufficient amount of antibodies to protect largepopulations is a challenge and currently has not been achieved. Theshelf life of antibodies is often relatively short (e.g., weeks ormonths), and accordingly freshly prepared batches of antibodies have tobe produced to replace the expiring antibodies.

Accordingly, there is a need for a cost effective and efficient way toprovide alternatives to current therapeutic agents. Further a needexists for alternative therapeutics that are easier to develop andproduce, have a longer shelf life, and bind as a single agent tomultiple targets on the same disease agent, as well as to differentdisease agents.

SUMMARY

An aspect of the invention provides a pharmaceutical composition fortreating a subject at risk for exposure to or exposed to at least onedisease agent, the pharmaceutical composition including: at least onerecombinant heteromultimeric neutralizing binding protein containing twoor more binding regions, such that the binding regions are not identicaland each binding region has affinity to specifically bind anon-overlapping portion of the disease agent, and the binding proteinneutralizes the disease agent thereby treating the subject for exposureto the disease agent. In a related embodiment, the binding proteinincludes at least one tag that is an epitope that is specificallyrecognized and bound by an antibody. For example the antibody isselected from: IgA, IgE, IgG, and IgM. In related embodiments, theanti-tag antibody that recognizes and specifically binds to the tagepitope, viz., the anti-tag antibody, is recombinantly produced andadministered to the subject. Alternatively, the anti-tag antibody isendogenously produced within the subject.

In various embodiments of the pharmaceutical composition, at least oneof the binding regions and/or the binding protein is selected from thegroup of: a single-chain antibody (scFv); a recombinant camelidheavy-chain-only antibody (VHH); a shark heavy-chain-only antibody(VNAR); a microprotein; a darpin; an anticalin; an adnectin; an aptamer;a Fv; a Fab; a Fab′; and a F(ab′)₂. In a related embodiment the bindingregions specifically bind to a surface, a portion, or an epitope of thedisease agent or molecular target. For example the epitope contains apeptide including an amino acid sequence, or is a carbohydrate sidechain of the peptide.

In a related embodiment of the pharmaceutical composition, the bindingregions or multimeric components of the heteromultimeric neutralizingbinding protein are associated non-covalently or covalently.

The binding protein in a related embodiment of pharmaceuticalcomposition includes a linker that separates the binding regions, suchthat the linker includes at least one selected from the group of: apeptide, a protein, a sugar, and a nucleic acid. In a relatedembodiment, the linker includes a carbohydrate. In a related embodiment,the linker does not affect ability of the binding protein to bind to thedisease agent or to a plurality of disease agents. For example, thelinker includes amino acid sequence GGGGS (SEQ ID NO: 54),GGGGSGGGGSGGGGS (SEQ ID NO: 55), a portion thereof, or substantiallyidentical.

The disease agent in various embodiments is at least one selected from:a virus, a cancer cell, a fungus, a bacterium, a parasite such as aprotozoan or an helmuth. Alternatively the disease agent is a pathogenicmolecule, which is a product of a disease agent, and is selected fromthe group of: a protein, a lipopolysaccharide, and a toxin. Thepathogenic molecule is secreted by or is produced by a disease-causingorganism, e.g., a pathogen or infectious agent such as a virus, abacterium, a prion, or a fungus. For example, the pathogenic moleculeresults from degradation of a disease-producing organism.

In various embodiments, the toxin is at least one selected from thegroup consisting of: an aflatoxin, a dinoflagellate toxin, a Botulinumtoxin, a Staphylococcal α-hemolysin, a Staphylococcal leukocidin, anaerolysin cytotoxic enterotoxin, a cholera toxin, a Bacillus cereushemolysis II, an Helicobacter pylori vacuolating toxin, a Bacillusanthracis toxin, a cholera toxin, an Escherichia coli serotype O157:H7toxin, an Escherichia coli serotype O104:H7 toxin, a lipopolysaccharideendotoxin, a Shiga toxin, a pertussis toxin, a Clostridium perfringensiota toxin, a Clostridium spiroforme toxin, a Clostridium difficiletoxin, Clostridium difficile toxin A, Clostridium difficile toxin B,Clostridium septicum α toxin, and Clostridium botulinum C2 toxin.

In various embodiments of the pharmaceutical composition, the at leastone disease agent includes a plurality of non-identical disease agents,and the binding protein binds to the plurality of the disease agents,thereby neutralizing the plurality of the non-identical disease agents.

The toxin in various embodiments of the pharmaceutical composition is aC. botulinum toxin, such that at least one of the binding regionsincludes a recombinant camelid heavy-chain-only antibody, and such thatthe pharmaceutical composition includes an amino acid sequence selectedfrom the group of:

(SEQ ID NO: 56)LVQVGGSLRLSCVVSGSDISGIAMGWYRQAPGKRREMVADIFSGGSTDYAGSVKGRFTISRDNAKKTSYLQMNNVKPEDTGVYYCRLYGSGDYWGQGTQVTVSSAHHSEDP; (SEQ ID NO: 57)LVHPGGSLRLSCAPSASLPSTPFNPFNNMVGWYRQAPGKQREMVASIGLRINYADSVKGRFTISRDNAKNTVDLQMDSLRPEDSATYYCHIEYTHYWGKGTLVTVSSEPKTPKPQ; and,(SEQ ID NO: 58)QVQLVESGGGLVQVGGSLRLSCVVSGSDISGIAMGWYRQAPGKRREMVADIFSGGSTDYAGSVKGRFTISRDNAKKTSYLQMNNVKPEDTGVYYCRLYGSGDYWGQGTQVTVSSAHHSEDPTSAIAGGGGSGGGGSGGGGSLQGQLQLVESGGGLVHPGGSLRLSCAPSASLPSTPFNPENNMVGWYRQAPGKQREMVASIGLRINYADSVKGRFTISRDNAKNTVDLQMDSLRPEDSATYYCHIEYTHYWGKGTLVTVSSEPKTPKPQ.

In a related embodiment of the pharmaceutical composition, the toxin isa C. difficile toxin A, such that the binding region includes arecombinant camelid heavy-chain-only antibody having an amino acidsequence selected from the group consisting of:

(SEQ ID NO: 59)QVQLVETGGLVQPGGSLRLSCAASGFTLDYSSIGWFRQAPGKEREGVSCISSSGDSTKYADSVKGRFTTSRDNAKNTVYLQMNSLKPDDTAVYYCAAFRATMCGVFPLSPYGKDDWGKGTLVTVSSEPKTPKPQP; (SEQ ID NO: 60)QLQLVETGGGLVQPGGSLRLSCAASGFTFSDYVMTWVRQAPGKGPEWIATINTDGSTMRDDSTKGRFTISRDNAKNTLYLQMTSLKPEDTALYYCARGRVISASAIRGAVRGPGTQVTVSSEPKTPKPQP; (SEQ ID NO: 61)QVQLVESGGGLVQPGGSLRLSCAASGFTLDYYAIGWERQAPGKEREGVSGISSVDGSTYYADSVRGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAADQSPIPIHYSRTYSGPYGMDYWGKGTLVTVSSAHHSEDP; (SEQ ID NO: 62)QLQLVESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVSGISFVDGSTYYADSVKGRFAISRGNAKNTVYLQMNSLKPEDTAVYYCAADQSSIPMHYSSTYSGPSGMDYWGKGTLVTVSSEPKTPKPQP; (SEQ ID NO: 63)QLQLVETGGGLVQAGGSLRLSCAASGRTLSNYPMGWERQAPGKEREEVAAIRRIADGTYYADSVKGRFTISRDNAWNTLYLQMNGLKPEDTAVYFCATGPGAFPGMVVTNPSAYPYWGQGTQVTVSSEPKTPKPQP; (SEQ ID NO: 64)QLQLVESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVSGISSSDGSTYYADSVKGRFTISRDNATNTVYLQMNSLKPEDTAVYYCAADQAAIPMHYSASYSGPRGMDYWGKGTLVTVSSEPKTPKPQP; (SEQ ID NO: 87)MSDKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPCKMIAPILDEIADEYQGKLTVAKLNIDQNPGTAPKYGIRGIPTLLLFKNGEVAATKVGALSKGQLKEFLDANLAGSGSGHMHHHHHHSSGLVPRGSGMKETAAAKFERQHMDSPDLGTDDDDKAMAISDPNSQVQLVESGGGLVQPGGSLRLSCEASGFTLDYYGIGWFRQPPGKEREAVSYISASARTILYADSVKGRFTISRDNAKNAVYLQMNSLKREDTAVYYCARRRFSASSVNRWLADDYDVWGRGTQVAVSSEPKTPKPQTSAIAGGGGSGGGGSGGGGSLQAMAAASQVQLVESGGGLVQTGGSLRLSCASSGSIAGFETVTWSRQAPGKSLQWVASMTKTNNEIYSDSVKGRFIISRDNAKNTVYLQMNSLKPEDTGVYFCKGPELRGQGIQVTVSSEPKTPKPQPARR; and, (SEQ ID NO: 95)MSDKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPCKMIAPILDEIADEYQGKLTVAKLNIDQNPGTAPKYGIRGIPTLLLFKNGEVAATKVGALSKGQLKEFLDANLAGSGSGHMHHHHHHSSGLVPRGSGMKETAAAKFERQHMDSPDLGTDDDDKAMAISDPNSQVQLVETGGLVQPGGSLRLSCAASGFTLDYSSIGWFRQAPGKEREGVSCISSSGDSTKYADSVKGRFTTSRDNAKNTVYLQMNSLKPDDTAVYYCAAFRATMCGVFPLSPYGKDDWGKGTLVTVSSEPKTPKPQPTSAIAGGGGSGGGGSGGGGSLQAMAAAQLQLVETGGGLVQPGGSLRLSCAASGFTFSDYVMTWVRQAPGKGPEWIATINTDGSTMRDDSTKGRFTISRDNAKNTLYLQMTSLKPEDTALYYCARGRVISASAIRGAVRGPGTQVTVSSEPKTPKPQPARQTSPSTVRLESRVRELEDRLEELRDELERAERRANEMSIQLDEC.

The toxin in various embodiments of the pharmaceutical composition is aC. difficile toxin B, such that at least one of the binding regions ofthe binding protein includes a recombinant camelid heavy-chain-onlyantibody having an amino acid sequence selected from the groupconsisting of:

(SEQ ID NO: 65)QVQLVESGGGLVQPGGSLRLSCAASGFSLDYYGIGWFRQAPGKERQEVSYISASAKTKLYSDSVKGRFTISRDNAKNAVYLEMNSLKREDTAVYYCARRRFDASASNRWLAADYDYWGQGTQVTVSSEPKTPKPQ; (SEQ ID NO: 66)QVQLVESGGGLVQAGGSLRLSCVSSERNPGINAMGWYRQAPGSQRELVAIWQTGGSLNYADSVKGRFTISRDNLKNTVYLQMNSLKPEDTAVYYCYLKKWRDQYWGQGTQVTVSSEP KTPKPQ;(SEQ ID NO: 67)QVQLVESGGGLVQPGGSLRLSCEASGFTLDYYGIGWFRQPPGKEREAVSYISASARTILYADSVKGRFTISRDNAKNAVYLQMNSLKREDTAVYYCARRRFSASSVNRWLADDYDVWGRGTQVAVSSEPKTPKPQ; (SEQ ID NO: 68)QVQLVESGGGLVQTGGSLRLSCASSGSIAGFETVTWSRQAPGKSLQWVASMTKTNNEIYSDSVKGRFIISRDNAKNTVYLQMNSLKPEDTGVYFCKGPELRGQGIQVTVSSEPKTPKPQ;(SEQ ID NO: 69)QVQLVESGGGLVEAGGSLRLSCVVTGSSFSTSTMAWYRQPPGKQREWVASFTSGGAIKYTDSVKGRFTMSRDNAKKMTYLQMENLKPEDTAVYYCALHNAVSGSSWGRGTQVTVSSE PKTPKPQ;(SEQ ID NO: 70)VQLVESGGGLVQAGGSLRLSCAASGLMFGAMTMGWYRQAPGKEREMVAYITAGGTESYSESVKGRFTISRINANNMVYLQMTNLKVEDTAVYYCNAHNFWRTSRNWGQGTQVTVS SEPKTPKP;(SEQ ID NO: 71)VQLVESGGGLVQAGDSLTLSCAASESTFNTFSMAWFRQAPGKEREYVAAFSRSGGTTNYADSVKGRATISTDNAKNTVYLHMNSLKPEDTAVYFCAADRPAGRAYFQSRSYNYWGQGTQVTVSSAHHSEDP; (SEQ ID NO: 72)VQLVESGGGSVQIGGSLRLSCVASGFTFSKNIMSWARQAPGKGLEWVSTISIGGAATSYADSVKGRFTISRDNANDTLYLQMNNLKPEDTAVYYCSRGPRTYINTASRGQGTQVTVSSEP KTPKP;(SEQ ID NO: 73)VQLVESGGGLVQAGGSLRLSCVGSGRNPGINAMGWYRQAPGSQRELVAVWQTGGSTNYADSVKGRFTISRDNLKNTVYLQMNSLKPEDTAVYYCYLKKWRDEYWGQGTQVTVSSAH HSEDP;(SEQ ID NO: 74)VQLVESGGGLVQAGESLRLSCVVSESIFRINTMGWYRQTPGKQREVVARITLRNSTTYADSVKGRFTISRDDAKNTLYLKMDSLKPEDTAVYYCHRYPLIFRNSPYWGQGTQVTVSSEPK TPKP;(SEQ ID NO: 75)VQLVESGGGLVQAGESLRLSCVVSESIFRINTMGWYRQTPGKQREVVARITLRNSTTYADSVKGRFTISRDDAKNTLYLKMDSLKPEDTAVYYCHRYPLIFRNSPYWGQGTQVTVSSEPK TP;(SEQ ID NO: 76); (SEQ ID NO: 87); and, SEQ ID NO: 95VQLVESGGGLVQAGGSLRLSCAAPGLTFTSYRMGWFRQAPGKEREYVAAITGAGATNYADSAKGRFTISKNNTASTVHLQMNSLKPEDTAVYYCAASNRAGGYWRASQYDYWGQGTQVTVSSAHHSEDP.

The toxin is a Shiga toxin in various embodiments of the pharmaceuticalcomposition, such that at least one of the binding regions includes arecombinant camelid heavy-chain-only antibody having an amino acidsequence selected from the group consisting of:

(SEQ ID NO: 77)QVQLVETGGGLAQAGDSLRLSCVEPGRTLDMYAMGWIRQAPGEEREFVASISGVGGSPRYADSVKGRFTISKDNTKSTIWLQMNSLKPEDTAVYYCAAGGDIYYGGSPQWRGQGTRVT VSSEPKTPKPQ;(SEQ ID NO: 78)QVQLVESGGGLVQAGGSLRLSCAASGRINGDYAMGWFRQAPGEEREFVAVNSWIGGSTYYTDSVKGRFTLSRDNAKNTLSLQMNSLKPEDTAVYYCAAGHYTDFPTYFKEYDYWGQGTQVTVSSEPKTPKPQ; (SEQ ID NO: 79)QVQLVETGGLVQAGGSLRLSCAASGVPFSDYTMAWFRQAPGKEREVVARITWRGGGPYYGNSGNGRFAISRDIAKSMVYLHMDSLKPEDTAVYYCAASRLRPALASMASDYDYWGQGTQVSVSSEPKTPKPQ; (SEQ ID NO: 80)QVQLVESGGGLVQPGESLRLSCVASASTFSTSLMGWVRQAPGKGLESVAEVRTTGGTFYAKSVAGRFTISRDNAKNTLYLQMNSLKAEDTGVYYCTAGAGPIATRYRGQGTQVTVSSA HHSEDP;(SEQ ID NO: 81)QVQLVESGGGLVQPGGSLKLSCAASGFTLADYVTVWFRQAPGKSREGVSCISSSRGTPNYADSVKGRATVSRNNANNTVYLQMNGLKPDDTAIYYCAAIRPARLRAYRECLSSQAEYDYWGQGTQVTVSSAHHSEDP; (SEQ ID NO: 82)QVQLVESGGGLVQPGGSLGLSCAMSGTTQDYSAVGWFRQAPGKEREGVSCISRSGRRTNYADSVRGRFTISRDNAKDTVYLWINSLKPDDTAVYYCAARKTDMSDPYYVGCNGMDYWGKGTLVTVSSAHHSEDP; (SEQ ID NO: 83)QVQLVESGGGLVQPGGSLTLSCTASGFTLNSYKIGWFRQAPGKEREGVSCINSGGNLRSVEGRFTISRDNTKNTVSLHMDSLKPEDTGVYHCAAAPALNVFSPCVLAPRYDYWGQGTQV TVSSAHHSEDP;(SEQ ID NO: 84)QVQLVESGGGLVQPGGSLRLSCAASGFTLGSYHIGWFRHPPGKEREGTSCLSSRGDYTKYAEAVKGRFTISRDNTKSTVYLQMNNLKPEDTGIYVCAAIRPVLSDSHCTLAARYNYWGQGTQVTVSSAHHSEDP; (SEQ ID NO: 85)QVQLVESGGGLVQPGGSLRLSCAALEFTLEDYAIAWFRQAPGKEREGVSCISKSGVTKYTDSVKGRFTVARDNAKSTVILQMNNLRPEDTAVYNCAAVRPVFVDSVCTLATRYTYWGEGTQVTVSSAHHSEDP; and (SEQ ID NO: 86)QVQLVETGGGLVQPGGSLKLSCAASEFTLDDYHIGWFRQAPGKEREGVSCINKRGDYINYKDSVKGRFTISRDGAKSTVFLQMNNLRPEDTAVYYCAAVNPVFPDSRCTLATRYTHWGQGTQVTVSSAHHSEDP.

The binding protein and/or at least one binding region in variousembodiments of the pharmaceutical composition includes an amino acidsequence that is substantially identical to at least one of SEQ ID NOs:56-87 and 95, wherein substantially identical is having at least 50%identity, 60% identity, at least 65% identity, at least 70% identity, atleast 75% identity, at least 80% identity, at least 85% identity, atleast 90% identity, or and at least 95% identity to the amino acidsequence of SEQ ID NOs: 56-87 and 95. Alternatively, the binding proteinand/or at least one binding region is encoded by at least one nucleotidesequence or the protein includes amino acid sequence selected from thegroup of SEQ ID NOs: 1-87 and 95, and substantially identical to any ofthese nucleotide sequences or amino acid sequences.

The pharmaceutical composition in various embodiments further includes asource of the binding protein. For example, the source of the bindingprotein is selected from the group of: a nucleic acid vector with a geneencoding the binding protein; a viral vector the binding protein; thebinding protein; and the binding protein expressed directly from nakednucleic acid. For example the vector encoding the binding protein is atleast one selected from an adenovirus, an adeno-associated virus, aherpesvirus, a poxvirus, and a lentivirus.

An aspect of the invention provides a method for treating a subject atrisk for exposure to or exposed to at least one disease agent, themethod including: contacting the subject with at least one recombinantheteromultimeric neutralizing binding protein including two or morebinding regions, such that the binding regions are not identical, andsuch that each binding region specifically binds a non-overlappingportion of the disease agent, so that the binding protein neutralizesthe disease agent thereby treating the subject for the exposure. In arelated embodiment, the binding protein includes at least one tag. Forexample the tag includes an epitope that synergistically increasesneutralization of the disease agent by the binding protein. In a relatedembodiment, the epitope is specifically recognized and bound by ananti-antibody, for example a phagocytic antibody or a clearance antibodyendogeneous produced by the subject. Alternatively, the anti-tagantibody is a recombinant antibody administered to the subject.

In various embodiments of the method, at least one binding region and/orthe binding protein is selected from the group of: a single-chainantibody (scFv); a recombinant camelid heavy-chain-only antibody (VHH);a shark heavy-chain-only antibody (VNAR); a microprotein; a darpin; ananticalin; an adnectin; an aptamer; a Fv; a Fab; a Fab′; and a F(ab′)₂.In various embodiments, the two or more binding regions included in thebinding protein are non-identical types of binding agents, for examplethe binding regions include non-identical VHHs, scFvs, VNARs,microproteins; darpins; an anticalins; adnectins; aptamers; Fvs; Fabs;Fab′s; a F(ab′)₂s, or combinations thereof.

The binding protein in various embodiments includes a linker thatseparates multimeric components of the binding regions. In relatedembodiments, the linker includes at least one selected from the groupof: a peptide, a protein, a sugar, or a nucleic acid. In relatedembodiments, the linker includes a single amino acid or a plurality ofamino acids. In a related embodiment, the linker includes amino acidsequence GGGGS (SEQ ID NO: 54) or a portion thereof. In a relatedembodiment, the linker includes amino acid sequence GGGGSGGGGSGGGGS (SEQID NO: 55) or a portion thereof or multiples thereof. The linker invarious embodiments stabilizes the binding protein and does not preventthe respective binding of the binding regions to the disease agent or toa plurality of disease agents.

In various embodiments of the method, the disease agent is at least oneselected from a virus, a cancer cell, a fungus, a bacterium, a parasiteand a product thereof such as a pathogenic molecule, a protein, alipopolysaccharide, and a toxin.

The toxin in various embodiments of the method is at least one selectedfrom the group of: Staphylococcal α-hemolysin, Staphylococcalleukocidin, aerolysin cytotoxic enterotoxin, a cholera toxin, Bacilluscereus hemolysis II, and Helicobacter pylori vacuolating toxin, Bacillusanthracis, cholera toxin, Escherichia coli serotype O157:H7, Escherichiacoli serotype O104:H7, lipopolysaccharide endotoxin, Shiga toxin,pertussis toxin, Clostridium perfringens iota toxin, Clostridiumspiroforme toxin, Botulinum neurotoxin, Clostridium difficile toxin A,Clostridium difficile toxin B, Clostridium septicum a toxin, andClostridium botulinum C2 toxin; and the bacterium is selected from thegroup consisting of: B. anthracis, B. cereus, C. botulinum, C.difficile, C. perfringens, C. spiroforme, and V. cholera.

The method further includes, prior contacting the subject with thebinding protein, engineering the binding regions and/or the bindingprotein. In certain embodiments of the method, the binding regions ofthe binding protein are specific to different classes/types of diseaseagents, such that each of the plurality of binding regions isnon-identical and is specific for a different bacteria, virus, fungus,cancer, and/or pathogenic molecule. For example a binding region isspecific for a virus and another binding region is specific for abacterium.

The method in various embodiments further includes observing and/ordetecting neutralization of the disease agent by the binding proteinsuch that the binding protein is effective for treating the subject andensuring survival of the subject. In a related embodiment, detectingneutralization by the binding protein includes identifying a reductionor remediation in at least one pathology symptom associated with thedisease agent. For example detecting and/or identifying neutralizationinvolves measuring an amount or a concentration of disease agent in thesubject or a sample from the subject, and comparing a control amount orconcentration prior to contacting, such that a decrease in the amount orthe concentration of the disease agent after contacting compared toprior to contacting is a positive indication that the subject istreated. For example, the sample is obtained from a cell, a tissue, or afluid from the subject.

In various embodiments of the method, the disease agent is a pluralityof disease agents, and the method involves prior to contacting,engineering the binding protein to bind to different domains of theplurality of the disease agents. For example the plurality of thedisease agents include at least two non-identical types of viruses,cancer cells, fungi, bacteria, parasites and products thereof suchpathogenic molecules, proteins, lipopolysaccharides, and toxins, andcombinations thereof.

The method in a related embodiment further includes, prior tocontacting, engineering at least one of the binding regions and/or thebinding protein with at least one amino acid sequence selected from thegroup of SEQ ID NOs: 56-87 and 95, and substantially identical.

In various embodiments of the method, contacting the subject with thebinding protein includes administering to the subject a source ofexpression of the binding protein, such that the source of theexpression of the binding protein is a nucleic acid encoding the bindingprotein, such that the nucleic acid includes at least one selected fromthe group consisting of: a naked nucleic acid vector, bacterial vector,and a viral vector.

An aspect of the invention provides a kit for treating a subject exposedto or at risk for exposure to a disease agent, the kit including: apharmaceutical composition for treating a subject at risk for exposureto or exposed to at least one disease agent, the pharmaceuticalcomposition including: at least one recombinant heteromultimericneutralizing binding protein comprising two or more binding regions,such that the binding regions are not identical and each binding regionhas affinity to specifically bind a non-overlapping portion of thedisease agent, and the binding protein neutralizes the disease agentthereby treating the subject for exposure to the disease agent; acontainer; and, instructions for use.

In various embodiments of the kit, at least one of the binding regionsand/or the binding protein is selected from the group of: a single-chainantibody (scFv); a recombinant camelid heavy-chain-only antibody (VHH);a shark heavy-chain-only antibody (VNAR); a microprotein; a darpin; ananticalin; an adnectin; an aptamer; a Fv; a Fab; a Fab′; and a F(ab′)₂.In related embodiments, at least one binding region and/or the bindingprotein includes a tag and/or a linker. In various embodiments the tagand/or the linker includes at least one selected from the group of: apeptide, a protein, a sugar, and a nucleic acid. In a related embodimentof the kit, the tag includes an amino acid sequence, for example SEQ IDNO:15, or a portion thereof. In related embodiments, the linker includesan amino acid sequence, for example the linker includes GGGGS (SEQ IDNO: 54), GGGGSGGGGSGGGGS (SEQ ID NO: 55), or a portion thereof.

The disease agent in various embodiments of the kit is selected from: avirus, a cancer cell, a fungus, a bacterium, a parasite and a productthereof such as a pathogenic molecule, a protein, a lipopolysaccharide,and a toxin. In a related embodiment of the kit, the disease agent is aplurality of disease agents. For example, the subject is exposed to aplurality of diseases, such as two or more diseases from any of viral,bacterial, fungal, and protozoal diseases. In various embodiments of thekit, the plurality of the disease agents includes two or more of a viralpathogen, and bacterial pathogens such as strains that produce aBotulinum toxin A, a Botulinum toxin B, a C. difficile toxin, or a Shigatoxin.

The toxin in various embodiments of the kit is at least one selectedfrom the group of: a Botulinum neurotoxin, Staphylococcal α-hemolysin,Staphylococcal leukocidin, aerolysin cytotoxic enterotoxin, a choleratoxin, Bacillus cereus hemolysis II, and Helicobacter pylori vacuolatingtoxin, Bacillus anthracis, cholera toxin, Escherichia coli serotypeO157:H7, Escherichia coli serotype O104:H7, lipopolysaccharideendotoxin, Shiga toxin, pertussis toxin, Clostridium perfringens iotatoxin, Clostridium spiroforme toxin, Clostridium difficile toxin A,Clostridium difficile toxin B, Clostridium septicum a toxin, andClostridium botulinum C2 toxin.

The toxin in various embodiments of the kit is a C. botulinum toxin,such that at least one binding region of the binding protein includes arecombinant camelid heavy-chain-only antibody, and such that thepharmaceutical composition includes an amino acid sequence selected fromthe group consisting of: SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58,and an amino acid sequence substantially identical to any of SEQ ID NOs:56-58 and portions thereof.

In a related embodiment of the kit, the toxin is a C. difficile toxin A,such that at least one binding region of the binding protein includes arecombinant camelid heavy-chain-only antibody having an amino acidsequence selected from the group consisting of: SEQ ID NO: 59, SEQ IDNO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQID NO: 87, SEQ ID NO: 95, and an amino acid sequence substantiallyidentical to any of SEQ ID NOs: 59-64, 87 and 95 and portions thereof.

The toxin in various embodiments is a C. difficile toxin B, such that atleast one binding region of the binding protein includes a recombinantcamelid heavy-chain-only antibody having an amino acid sequence selectedfrom the group consisting of: SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO:67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ IDNO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQID NO: 87, SEQ ID NO: 95, and an amino acid sequence substantiallyidentical to any of SEQ ID NOs: 66-76, 87 and 95 and portions thereof.

The toxin in various embodiments of the kit is a Shiga toxin, such thatat least one binding region of the binding protein includes arecombinant camelid heavy-chain-only antibody having an amino acidsequence selected from the group consisting of: SEQ ID NO: 77, SEQ IDNO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, and an aminoacid sequence substantially identical to any of SEQ ID NOs: 77-86 andportions thereof.

An aspect of the invention provides a composition including at least oneamino acid sequence selected from the group of: SEQ ID NO: 56, SEQ IDNO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66,SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO:71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ IDNO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85,SEQ ID NO: 86; SEQ ID NO: 87, SEQ ID NO: 95, and substantiallyidentical, wherein substantially identical is an amino acid sequencehaving at least 50% identity, at least 60% identity, at least 65%identity, at least 70% identity, at least 75% identity, at least 80%identity, at least 85% identity, at least 90% identity, at least 95%identity, or at least 99% identity to any of the amino acid sequence ofSEQ ID NOs: 56-87 and 95 and portions thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 panels A-E are nucleotide sequences of scFv#2 (SEQ ID NO: 1),scFv#3 (SEQ ID NO: 3), scFv#7 (SEQ ID NO: 5), scFv#8 (SEQ ID NO: 7),scFv#21 (SEQ ID NO: 9), scFv#E (SEQ ID NO: 11), and amino acid sequencesof scFv#2 (SEQ ID NO: 2), scFv#3 (SEQ ID NO: 4), scFv#7 (SEQ ID NO: 6),scFv#8 (SEQ ID NO: 8), scFv#21 (SEQ ID NO: 10), scFv#E (SEQ ID NO: 12).

FIG. 2 is the nucleotide sequence of scFv#7-2E (SEQ ID NO: 13) and theamino acid sequence of scFv#7-2E (SEQ ID NO: 14).

FIG. 3 panels A-C are the nucleotide sequences of BoNT/A holotoxinbinding VHHs including JDA-D12 (SEQ ID NO: 19), JDQ-A5 (SEQ ID NO: 21),JDQ-B5 (SEQ ID NO: 23), JDQ-C2 (SEQ ID NO: 25), JDQ-F12 (SEQ ID NO: 27),JDQ-G5 (SEQ ID NO: 29), JDQ-H7 (SEQ ID NO: 31), and BoNT/B holotoxinbinding VHHs including JEQ-A5 (SEQ ID NO: 33), JEQ-H11 (SEQ ID NO: 35).The figures also show the corresponding amino acid sequences of BoNT/Aholotoxin binding VHHs including JDA-D12 (SEQ ID NO: 20), JDQ-A5 (SEQ IDNO: 22), JDQ-B5 (SEQ ID NO: 24), JDQ-C2 (SEQ ID NO: 26), JDQ-F12 (SEQ IDNO: 28), JDQ-G5 (SEQ ID NO: 30), JDQ-H7 (SEQ ID NO: 32), and BoNT/Bholotoxin binding VHHs including JEQ-A5 (SEQ ID NO: 34), JEQ-H11 (SEQ IDNO: 36).

FIG. 4 panel A is a list of nucleotide sequences of VHHs identified asBoNT/A binders that were experimentally shown to bind to the sameepitope. The VHH sequences are DQ-B5 (SEQ ID NO: 23), JDQ-E9 (SEQ ID NO:37), JDQ-B2 (SEQ ID NO: 39), JDQ-05 (SEQ ID NO: 41), and JDQ-F9 (SEQ IDNO: 43).

FIG. 4 panel B is a list of corresponding VHH amino acid sequences,JDQ-B5 (SEQ ID NO: 24), JDQ-E9 (SEQ ID NO: 38), JDQ-B2 (SEQ ID NO: 40),JDQ-05 (SEQ ID NO: 42), and JDQ-F9 (SEQ ID NO: 44), of the nucleic acidsequences in FIG. 4 panel A.

FIG. 5 is a schematic drawing of a phylogenetic tree comparing thehomology between BoNT/A binding VHHs within the JDQ-B5 competition group(which compete for binding, thus bind the same epitope) in comparison tocontrol alpaca VHHs.

FIG. 6 is a schematic drawing of binding agent VHHs that are produced indifferent formats including formats in which the binding agents arefused to one or more E-tags or as fusion proteins.

FIG. 7 is a drawing of a single-tagged heterodimeric binding protein(exemplary VHHs) binding to the disease agent, a toxin, and leading todecoration of the toxin with two anti-tag monoclonal antibodies (mAbs).

FIG. 8 is a drawing of a double-tagged binding protein (here shown areVHHs) a heterodimeric binding to the disease agent, toxin, and leadingto decoration of the toxin with four anti-tag mAbs.

FIG. 9 panels A-B are a set of Meyer-Kaplan survival plots thatdouble-tagged heterodimer E/H7/B5/E and the anti-tag mAb completelyprotected subjects from 1.000-fold and 1.000-fold the median lethal doseof a Botulinum neurotoxin serotype A toxin.

FIG. 9 panel A is a Meyer-Kaplan survival plot showing percent (%) ofmice surviving over a period of time (days) after receiving 1.000-foldthe median lethal dose (LD₅₀) of a Botulinum neurotoxin serotype A(BoNT/A) and each of combinations of the following binding agents: H7and B5 VHH heterodimer with a single epitopic tag (tag or E-tag) and ananti-E-tag mAb (H7/B5/E+anti-E mAb); H7 and B5 VHH monomers each with anE-tag and an anti-E-tag mAb (H7/E+B5/E+anti-E mAb); H7 and B5 VHHheterodimer with two E-tags and an anti-E-tag mAb (E/H7/B5/E+anti-E mAb)and a control (the toxin alone). The data show that administration ofheterodimer E/H7/B5/E and anti-E mAb resulted in survival of subjectsfor seven days.

FIG. 9 panel is a Meyer-Kaplan survival plot showing percent (%) ofsubjects surviving over a period of time (days) after receiving10.000-fold the LD₅₀ of a Botulinum neurotoxin (BoNT) and H7 and B5 VHHheterodimer with two E-tags and an anti-E-tag mAb (E/H7/B5/E+anti-E mAb)and a control (the toxin alone). Remarkably, 100% of the mice survived a10,000 LD₅₀ challenge of BoNT/A when administered the double-taggedheterodimer and the anti-tag mAb.

FIG. 10 panels A-B are nucleotide sequences and amino acid sequences ofrecombinant BoNT/A holotoxin binding VHHs: thioredoxin/JDQ-H7(H7)/E-tag(SEQ ID NO: 45), thioredoxin/JDQ-B5(B5)/E-tag (SEQ ID NO: 47),thioredoxin/H7/flexible spacer (fs)/B5/E-tag (SEQ ID NO: 49), andthioredoxin/E-tag/H7/fs/B5/E-tag (SEQ ID NO: 51). The correspondingamino acid sequences of the VHHs including amino acid sequences forthioredoxin/H7/E-tag (SEQ ID NO: 46), thioredoxin/B5/E-tag (SEQ ID NO:48), thioredoxin/H7/fs/B5/E-tag (SEQ ID NO: 50),thioredoxin/E-tag/H7/fs/B5/E-tag (SEQ ID NO: 52), and thioredoxin (SEQID NO: 53) are shown.

FIG. 11 panels A-B are Meyer-Kaplan survival plots showing percentsurvival (% survival, ordinate) of subjects as a function of time indays (abscissa) following contact with BoNT/A and later time (1.5 hoursor three hours later) administered VHH binding/neutralizing agents.Subjects (five per group) were intravenously exposed to 10 LD₅₀(ten-fold LD₅₀) of BoNT/A, and then later administered either: a mixtureof 1 μg ciA-H7 monomer (SEQ ID NO: 32) and 1 μg of ciA-B5 monomer (SEQID NO: 24); H7/B5 heterodimeric protein (SEQ ID NO: 58); a sheepantitoxin serum; or control (no binding agent). Data show that the H7/B5heterodimer was effective as an antitoxin neutralizing agent andprotected subjects from the lethal challenge of BoNT/A.

FIG. 11 panel A shows percent survival for subjects exposed to ten-foldLD₅₀ of BoNT/A then administered 1.5 hours later either a mixture of H7and B5 monomers; H7/B5 heterodimer; a sheep serum antitoxin; or controltoxin only (no agents).

FIG. 11 panel B shows percent survival for subjects exposed to ten-foldLD₅₀ of BoNT/A then administered three hours later either a mixture ofH7 and B5 monomers; H7/B5 heterodimer; a sheep serum antitoxin; orcontrol toxin only (no agents).

FIG. 12 panels A-C are line graphs showing that VHH monomers and VHHheterodimers neutralized C. difficile toxin b (TcdB) and protectedsubjects from death caused by exposure to TcdB.

FIG. 12 panel A is a line graph showing that VHH monomers neutralized C.difficile toxin B (TcdAB) and protected cells from the toxin. Thepercent CT26 cells affected by TcdB (% affected; ordinate) is shown as afunction of concentration (0.003 nM, 0.03 nM, 0.3 nM, 3 nM, 30 nM, 300nM, or 3000 nM) of administered VHH monomers: 5D (circle), 2D (square),or E3 (light upward facing triange). Control cells were administeredtoxin only (TcdB; dark downward facing triangle). Strength ofneutralizing VHH activity was observed in the order 5D as strongestfollowed by E3 and 2D.

FIG. 12 panel B is a line graph showing percent of cells affected byTcdB (% affected; ordinate) as a function of concentration ofadministered mixture of 5D and E3 monomers, 5D/E3 heterodimer (VHH;abscissa), or a toxin only control. It was observed that the 5D/E3 VHHheterodimer (squares) was about ten-fold more potent as toxinneutralizing agent than the mixture of 5D monomer and E3 monomer(triangles).

FIG. 12 panel C is a Meyer-Kaplan survival plot of a C. difficileinfection model showing percent mouse survival (ordinate) as a functionof time (hours post challenge, abscissa) of subjects co-administeredtoxin and VHH neutralizing agents. Subjects were co-administered alethal dose of TcdB with: a mixture of 10 μg of 5D monomers and E3monomers (5 μg of each monomer per mouse; dashed line, blue); a mixtureof 1 μg of 5D monomers and E3 monomers (500 ng of each monomer permouse; thick solid line, blue), 5D/E3 heterodimer (250 ng per mouse;light solid line, red), or phosphate-buffered saline (PBS; thin solidline, black). Percent survival was calculated for each group ofsubjects.

FIG. 13 panels A-C are amino acid sequences for VHH monomers and VHHheterodimers designed to specifically bind epitopes of botulism toxinsserotype A (BoNT/A) and serotype B (BoNT/B). Each VHH was purified fromE. coli as a thioredoxin fusion protein having a singlecarboxyl-terminal epitopic tag (tag or E-tag).

FIG. 13 panel A is a set of amino acid sequences of VHH monomers thatspecifically recognize and bind to epitopes on BoNT/A (ciA-A5, ciA-B5,ciA-D12, ciA-F12, ciA-G5, and ciA-H7) and epitopes of BoNT/B (ciB-A11,ciB-B5, ciB-B9, and ciB-H11). The sequences are aligned to showhomology. Dashed regions of the amino acid sequences are spaces insertedto align the amino acid regions.

FIG. 13 panel B is a set of amino acid sequences of VHH monomers(ciA-D1, ciA-H5, and ciA-H11) that bind specifically to the same epitopeof BoNT/B as ciA-H7.

FIG. 13 panel C shows amino acid sequences for double-tagged VHHheterodimers, ciA-H7/ciA-B5(2E) and ciA-F12/CiA-D12(2E), thatspecifically bind BoNT/A.

FIG. 14 panels A-B are photographs of sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) analyses of VHH monomersand VHH heterodimers.

FIG. 14 panel A shows SDS-PAGE analysis of the tagged (E) VHH monomersciA-D1, ciA-H4, ciA-H11, ciA-A5, ciA-C2, ciA-D12, ciA-F12, ciA-G5, andciA-H7.

FIG. 14 panel B is a SDS-PAGE analysis of single- or double-tagged VHHheterodimers including: ciA-H7/ciA-B5 singly tagged on ciA-B5 (leftchannel); double tagged ciA-H7/ciA-B5 having a tag on both ciA/H7 andciA-B5 (second channel from left), ciA-F12/ciA-D12 singly tagged onciA-B5 (third channel from the left); double tagged ciA-F12/ciA-D12having a tag on both ciA/F12 and ciA-D12 (fourth channel from left),double tagged ciA-A11/ciA-B5 having a tag on both ciA/A11 and ciA-B5(right channel).

FIG. 15 are photographs of Western blots showing ability of VHH monomersto prevent BoNT/A from cleavaging of synaptosomal-associated protein 25(SNAP25) in primary neurons in culture.

FIG. 16 panels A-C is a set of drawings and Meyer-Kaplan survival plotsshowing that mouse subjects administered each of a set of mixtures ofVHH monomers in combination with anti-tag clearing antibody wereprotected from BoNT/A.

FIG. 16 panel A (top) is a drawing of a BoNT/A bound to two differenttagged binding protein monomers that are each specifically bound by ananti-tag antibody. FIG. 16 panel A (bottom) is a set of graphs showingpercent of survival (% survival, ordinate) as a function of time (days,abscissa) of subjects co-administered 100-fold (FIG. 16 panel A bottomleft graph) or 1,000-fold (FIG. 16 panel A bottom right graph) the LD₅₀of a BoNT/A and combinations of VHH monomers (ciA-D12 and ciA-F12) withor without anti-tag clearing antibody (+αE and −αE respectively). Themixture of VHH monomer B5, VHH monomer H7 and anti-tag clearing antibodyprotected subjects from the 100-fold LD50 of toxin.

FIG. 16 panel B (top) is a drawing of a BoNT/A bound to three differentmonomeric tagged binding protein each specifically bound by an anti-tagantibody. FIG. 16 panel B (bottom) is a set of graphs showing percentsurvival on the ordinate as a function of time (days, abscissa) ofsubjects co-administered 1,000-fold BoNT/A LD₅₀ (FIG. 16 panel B bottomleft graph) or 10,000-fold BoNT/A LD₅₀ (FIG. 16 panel bottom B rightgraph), and combinations of three VHH monomers with or without anti-tagclearing antibody (+αE and −αE respectively).

FIG. 16 panel C (top) is a drawing of a BoNT/A bound to four differenttagged binding protein monomers that are each specifically bound by ananti-tag antibody. FIG. 16 panel C (bottom) is a set of graphs showingpercent survival, ordinate, of subjects as a function of time (days,abscissa) of subjects co-administered 1,000-fold BoNT/A LD₅₀ (FIG. 16panel C bottom left graph) or 10,000-fold BoNT/A LD₅₀ (FIG. 16 panel Cbottom right graph), and a mixture of ciA-B5, ciA-H7, ciA-D12 andciA-F12 VHH monomers with (+αE) or without (−αE) anti-tag clearingantibody.

FIG. 17 are graphs showing percent survival, ordinate, of subjects as afunction of time (days, abscissa) of subjects co-administered 1,000-foldBoNT/A LD₅₀ (FIG. 17 left graph) or 10,000-fold BoNT/A LD₅₀ (FIG. 17right graph), and mixtures of VHH monomers and anti-tag clearingantibody (αE). Control subjects received toxin only. Unless indicatedotherwise, an asterisk (*) in FIGS. 17-24 indicates that the subjectsadministered the VHH monomer or multimer displayed no symptoms of toxinexposure.

FIG. 18 panels A-B are a table showing affinity binding data for VHHsand a set of line graphs showing improved protection of subjects fromvery large doses of BoNT/A following administration of each of sets ofmixtures of VHH monomers with strong affinity for BoNT/A and clearingantibody.

FIG. 18 panel A is a table showing binding affinities (Kd) determined bysurface plasmon resonance (SPR) analysis of each of VHH monomers ciA-H7,ciA-D1, ciA-H4, and ciA-H11. SPR analysis was used to determine thebinding affinities to epitope A1 of BoNT/A for each VHH monomer. H7 hasthe greatest affinity and H11 the least affinity.

FIG. 18 panel B is a set of graphs showing percent survival on theordinate of subjects as a function of time (days, abscissa) followingco-administration of BoNT/A at 100-fold (FIG. 18 panel B left graph) or1,000-fold (FIG. 18 panel B right graph) the LD₅₀, and a mixture of twoVHH monomers (B5+C2) or a mixture of three VHH monomers with anti-tagclearing antibody: B5+C2+H11; B5+C2+H7; B5+C2+D1; or B5+C2+112.

FIG. 19 panels A-B are drawings and graphs showing that administeringheterodimers composed of neutralizing VHH components resulted in greaterantitoxin efficacy than heterodimers composed of non-neutralizing VHHs,and that presence of two or more F-tags within the VHH heterodimersfurther increased the antitoxin efficacy.

FIG. 19 panel A (top) is a drawing of a BoNT/A bound to two differenttagged heterodimer binding proteins that are each specifically bound byan anti-tag antibody. FIG. 19 panel A (bottom) is a set of graphsshowing percent survival on the ordinate of subjects as a function oftime (days, abscissa) after co-administration of 1,000-fold (FIG. 19panel A bottom left graph) or 10,000-fold (FIG. 19 panel A bottom rightgraph) the BoNT/A LD₅₀, and a VHH heterodimer composition with (+αE) orwithout (−αE) anti-tag clearing antibody. The tagged VHH heterodimercomposition was either composed of neutralizing VHHs ciA-H7 and ciA-B5(H7/B5), or of non-neutralizing VHHs ciA-D12 and ciA-F12 (D12/F12). Datashow that subjects administered the heterodimer composition containingneutralizing VHHs ciA-B5 and ciA-4-17 survived longer than subjectsadministered the heterodimer composition containing non-neutralizingVHHs ciA-D12 and ciA-F12. Subjects administered clearing anti-tagantibodies generally survived longer than subjects not administeredclearing-tag antibodies.

FIG. 19 panel B (top) is a drawing of a BoNT/A bound to two differentdouble-tagged heterodimer binding proteins that are each specificallybound by two anti-tag antibodies. FIG. 19 panel B (bottom) is a set ofgraphs showing percent survival, ordinate, of subjects as a function oftime (days, abscissa) after co-administration of an amount of BoNT/A1,000-fold (FIG. 19 panel B bottom left graph) or 10,000-fold (FIG. 19panel B bottom right graph) the LD₅₀, and double tagged VHH heterodimerswith (+αE) or without (−αE) anti-tag clearing antibody. Subjectsadministered neutralizing ciA-B5/ciA-H7 heterodimer survived longer thansubjects administered non-neutralizing ciA-D12/ciA-F12 heterodimer. Datashow that all subjects administered double-tagged ciA-B5/ciA-H7heterodimers and anti-tag clearing antibody survived exposure to1,000-fold (FIG. 19 panel B bottom left graph) or 10,000-fold the LD₅₀of BoNT/A (FIG. 19 panel B bottom right graph).

FIG. 20 is a set of graphs showing percent survival on the ordinate ofsubjects as a function of time (days, abscissa) after co-administrationof 100-fold (FIG. 20 left graph) or 1,000-fold (FIG. 20 right graph)BoNT/A LD₅₀, and multi-tagged VHH heterodimers with anti-tag clearingantibody. The ciA-D12/ciA-F12 heterodimer protein contained either onetag (1e), two tags (2e), three tags (3e), or control no tag. Subjects(five mice per group) were administered 20 μg of the heterodimercomposition or the mixture of ciA-D12 and ciA-F12 monomers (20 μg ofeach monomer). Control subjects were administered neither monomer norheterodimer. Each subject received 60 picomoles of anti-E-tag clearingantibody. Data show that subjects administered ciA-D12/ciA-F12heterodimers having either one tag or two tags survived (100% survival)the challenge of 100-fold the LD₅₀ of BoNT/A (FIG. 20 left graph).Subjects receiving 1,000-fold the LD₅₀ of BoNT/A and ciA-D12/ciA-F12heterodimers with clearing antibody died within one day followingchallenge with independent of number of tags (FIG. 20 right graph).

FIG. 21 is a set of graphs showing percent survival, ordinate, ofsubjects treated with different amounts of anti-tag clearing antibody asa function of time (days, abscissa) after exposure to BoNT/A 100-fold(FIG. 21 left graph) or 1,000-fold (FIG. 21 right graph) the LD₅₀ and todouble tagged ciA-D12/ciA-F12 heterodimer (20 picomoles). Anti-tagclearing antibody was administered at: 20 picomoles, 40 picomoles, 60picomoles, 120 picomoles, or control (none). Control subjects receivedtoxin only (no agents). Data show improved antitoxin efficacy insubjects co-administered amounts (40, 60 or 120 picomoles) increasedanti-tag clearing antibody compared to 20 picomoles.

FIG. 22 is a graph showing percent survival, ordinate, of subjectstreated with different doses of double tagged neutralizing ciA-B5/ciA-H7heterodimers as a function of time (days, abscissa) for subjectsco-administered 1,000-fold BoNT/A LD₅₀, and anti-tag clearing antibody.Heterodimer ciA-B5/ciA-H7 was administered in doses of: 1.5 picomoles,4.4 picomoles, 13 picomoles, or 40 picomoles. Control subjects receivedtoxin only (no agents). Data show complete survival after seven days ofsubjects receiving amounts of 13 picomoles or 40 picomoles double taggedneutralizing ciA-B5/ciA-H7 heterodimer, such that than 13 picomolesprotected subjects fully from 1,000-fold BoNT/A LD₅₀, compared to 1.5picomoles or 4.4 picomoles (no survival after one day).

FIG. 23 panels A-B are graphs showing percent survival, ordinate, aftersubjects were exposed to ten-fold BoNT/A LD₅₀ and were administereddouble-tagged heterodimer and anti-tag clearing antibody of subjects asa function of time (days, abscissa). Administration of heterodimer aftertoxin exposure was observed to have protected subjects from symptoms anddeath caused by exposure to ten-fold BoNT/A LD₅₀.

FIG. 23 panel A is a set of graphs showing percent survival of subjectsas a function administration of: double tagged ciA-D12/ciA-F12heterodimer with anti-tag clearing antibody (+αE), double taggedciA-D12/ciA-F12 heterodimer without anti-tag clearing antibody (−αE), asheep serum antitoxin, or toxin only control (no agents). Prior toadministration of heterodimer, subjects were exposed 1.5 hours (FIG. 23panel A left graph) or three hours (FIG. 23 panel A right graph) toten-fold BoNT/A LD₅₀. Data show 100% survival of subjects administeredciA-D12/ciA-F12 heterodimer and anti-tag antibody after 1.5 hours.Survival of subjects administered ciA-D12/ciA-F12 heterodimer wascomparable to that in subjects administered sheep serum antitoxin.

FIG. 23 panel B is a set of graphs showing percent survival of subjectsas a function administration of: double tagged ciA-B5 and ciA-F17heterodimer with anti-tag clearing antibody (+αE), or with double taggedciA-B5/ciA-H7 heterodimer without anti-tag clearing antibody (−αE), orwith a sheep serum antitoxin, or toxin only control (no agents). Priorto treatment with heterodimer, subjects were exposed to ten-fold BoNT/ALD₅₀ either 1.5 hours (FIG. 23 panel B left graph) or three hours (FIG.23 panel B right graph). Data show that subjects administeredciA-B5/ciA-H7 heterodimer with or without anti-E tag antibody survivedlonger than subjects administered sheep serum antitoxin. Survival ofsubjects administered ciA-B5/ciA-H7 heterodimer was greater thansubjects administered sheep serum antitoxin.

FIG. 24 panels A-B are line graphs showing that subjects administeredciA-A11/ciA-B5 heterodimers with anti-tag clearing antibody wereprotected from BoNT/B exposure.

FIG. 24 panel A is a graph showing survival on the ordinate as afunction of time (days, abscissa) co-administration of 1,000-fold (FIG.24 panel A left graph) or 10,000-fold (FIG. 24 panel A right graph)BoNT/B LD₅₀ and a combination of ciB-All and ciB-B5 heterodimer with(+αE) or without (−αE) anti-tag clearing antibody, or toxin only control(no agents). Data show that subjects administered ciA-A11/ciA-B5heterodimer and anti-E-tag clearing antibody survived and were protectedlonger from BoNT/A than control subjects administered no agents and noanti-E tag antibody.

FIG. 24 panel B is a set of graphs showing subject survival (ordinate)as a function of time, abscissa, after administration of: double taggedciB-A11 and ciB-B5 heterodimer and anti-tag clearing antibody (+αE), ordouble tagged ciB-A11 and ciB-B5 heterodimer without anti-tag clearingantibody (−αE), a sheep serum antitoxin, or toxin only control.Following 1.5 hours (FIG. 24 panel B left graph) or three hours (FIG. 24panel B right graph) exposure to ten-fold BoNT/B LD₅₀, the subjects wereadministered the heterodimer. A greater percentage of subjectsadministered ciB-A11 and ciB-B5 heterodimer survived exposure to BoNT/Bthan subjects administered sheep serum antitoxin.

FIG. 25 is a line graph of percent of cells affected by C. difficiletoxin A (TcdA) and protection of cells from the toxin by VHH monomers.The percent CT26 cells affected by TcdA (% affected; ordinate) is shownas a function of concentration (0.1 nM, 0.48 nM, 2.4 nM, 12 nM, 60 nM,or 300 nM) of each administered VHH monomer: A3H (circle), A11G (lightsquare); AC1 (upward dark empty triangle), AH1 (upward light triangle),AH3 (downward triangle), or AA6 (dark empty square). Control cells wereadministered toxin only (TcdA; dark downward triangle). Strength ofneutralizing VHH activity was observed in the following order: AA6 asstrongest, then AH3, AC1, AE1, A11G, and A3H as weakest.

FIG. 26 is a line graph showing percent CT26 cells affected after 24hours of TcdA exposure (ordinate) as a function of concentrationadministered (abscissa: 0.03 ng/mL, 0.1 ng/mL, 1 ng/mL, 3 ng/mL, 10ng/mL, 30 ng/mL, 100 ng/mL, 300 ng/mL, or 1000 ng/mL), or toxin onlycontrol (TcdA; vertical line). Agents administered were: VHH monomer AH3(AH3, diamond), VHH monomer AA6 (AA6, square), a mixture of VHH monomersAH3 and AA6 (AH3+AA6, triangle), VHH heterodimer of AH3 and AA6(AH3/AA6, -x-); or a homodimer of heterodimer (tetramer) containing AH3and AA6 using a dimerizer sequence oAgB (AH3/AA6/oAgB, stars; SEQ ID NO:95). Control cells were treated with medium only. Percent cell roundingwas analyzed using a phase contrast microscope. It was observed that thehomodimer of the heterodimer containing AH3 and AA6 resulted in thestrongest TcdA neutralization.

FIG. 27 is a set of line graphs showing percent affected CT26 cellsexposed to toxin (ordinate) and then contacted with VHH heterodimer of5D and AA6 (FIG. 27 left graph) or with heterodimer of 5D and AH3 (FIG.27 right graph) as a function of concentration of VHH (abscissa: 0.01nM, 0.03 nM, 0.1 nM, 0.3 nM, 1 nM, 3 nM, 10 nM, or 30 nM). CT26 cellswere exposed overnight to TcdA (2 ng/mL; diamond) or TcdB (0.1 ng/mL;square), and then treated with either heterodimer 5D/AA6 (FIG. 27 leftgraph) or heterodimer 5D/AH3 (FIG. 27 right graph). Each heterodimerincluded a VHH monomer (5D) that neutralized TcdB, and a VHH monomer(AA6 or AH3) that neutralized TcdA. Data show that the treatment waseffective to protect cells from both toxins.

FIG. 28 panels A-C are a drawing, a line graph and a bar graph showingthat a VHH heterodimer of 5D and AA6 protected mouse subjects from TcdAand TcdB in an oral C. difficile spore challenge model.

FIG. 28 panel A is a protocol for a clinically relevant murine C.difficile infection model. Administration of VHH is given after a sporechallenge.

FIG. 28 panel B shows percent survival (ordinate) as a function of timefollowing spore challenge (abscissa) for subjects administered 5D/AA6heterodimer as described in FIG. 28 panel A. Data show that after thespore challenge, 90% of 5D/AA6 heterodimer contacted-subjects survived,and all control subjects not administered 5D/AA6 heterodimer or otheragent died within two days.

FIG. 28 panel C showing percent diarrhea (ordinate) as a function oftime following spore challenge (abscissa) for subjects administered5D/AA6 heterodimer (5D/AA6 TrxA; left bar), or control PBS (right bar)as described in FIG. 28 panel A. Data show that 5D/AA6 heterodimeradministered-subjects were five-fold less likely to display symptoms ofdiarrhea than control untreated subjects.

DETAILED DESCRIPTION

The presence of toxins in the circulation is the cause of a wide varietyof human and animal illnesses. Antitoxins are therapeutic agents thatprevent toxin infection or reduce further development of negativesymptoms in patients that have been exposed to a toxin (a processreferred to as “intoxication”). Typically, antitoxins are antiseraobtained from large animals (e.g., sheep, horse, and pig) that wereimmunized with inactivated or non-functional toxin. More recently,antitoxin therapies have been developed using combinations of antitoxinmonoclonal antibodies including yeast-displayed single-chain variablefragment antibodies generated from vaccinated humans or mice. SeeNowakowski et al. 2002. Proc Natl Acad Sci USA 99: 11346-11350;Mukherjee et al. 2002. Infect Immun 70: 612-619; Mohamed et al. 2005Infect Immun 73: 795-802; Walker, K. 2010 Interscience Conference onAntimicrobial Agents and Chemotherapy—50th Annual Meeting—Research onPromising New Agents: Part 1. IDrugs 13: 743-745. Antisera andmonoclonal antibodies can be difficult to produce economically at scale,usually requiring long development times and resulting in problematicquality control, shelf-life and safety issues. New therapeuticstrategies to develop and prepare antitoxins are needed.

Antitoxins function through two key mechanisms neutralization of toxinfunction and clearance of the toxin from the body. Toxin neutralizationoccurs through biochemical processes including inhibition of enzymaticactivity and prevention of binding to cellular receptors. Antibodymediated serum clearance occurs subsequent to the binding of multipleantibodies to the target antigen (Daeron M. 1997 Annu Rev Immunol 15:203-234; Davies et al. 2002 Arthritis Rheum 46: 1028-1038; Johansson etal. 1996 Hepatology 24: 169-175; and Lovdal et al. 2000 J Cell Sci 113(Pt 18): 3255-3266). Multimeric antibody decoration of the target isnecessary to permit binding to low affinity Fc receptors (Davies et al.2002 Arthritis Rheum 46: 1028-1038 and Lovdal et al. 2000 J Cell Sci 113(Pt 18): 3255-3266). Without being limited by any particular theory ormechanism of action, it is here envisioned that an ideal antitoxintherapeutic would both promote toxin neutralization to immediately blockfurther toxin activity and also accelerate toxin clearance to eliminatefuture pathology if neutralization becomes reversed.

Effective clearance of botulinum neurotoxin (BoNT), a National Instituteof Allergy and Infectious Diseases (NIAID) Category A priority pathogen,is believed by some researchers to require three or more antibodiesbound to the toxin. Nowakowski et al. 2002. (Proc Natl Acad Sci USA 99:11346-11350) determined that effective protection of mice against highdose challenge of BoNT serotype A (BoNT/A) required co-administration ofthree antitoxin monoclonal antibodies, and that all three antibodiespresumably promoted clearance. Data have shown that administration of apool of three or more small binding agents, each produced with a commonepitopic tag, reduced serum levels of a toxin when co-administered withan anti-tag monoclonal antibody (Shoemaker et al. U.S. publishedapplication 2010/0278830 A1 published Nov. 4, 2010 and Sepulveda et al.2009 Infect Immun 78: 756-763, each of which is incorporated herein inits entirety). The tagged binding agents directed the binding ofanti-tag monoclonal antibody to multiple sites on the toxin, thusindirectly decorating the toxin with antibody Fe domains and leading toits clearance through the liver.

Pools of scFv domain binding agents with specificity for BoNT/A and eachcontaining a common epitopic tag (E-tag), had been shown to be effectivefor decorating the botulinum toxin with multiple anti-tag antibodies(Shoemaker et al. U.S. utility patent publication number 2010/0278830published Nov. 4, 2010 and U.S. continuation-in-part patent publicationnumber 2011/0129474 published Jun. 2, 2011, each of which isincorporated herein by reference in its entirety). Data showed that theadministration of binding agents and clearance antibodies to subjectsresulted in clearance via the liver with an efficacy in mouse assaysequivalent to conventional polyclonal antitoxin sera. Ibid. andSepulveda et al. 2009 Infect Immun 78: 756-763. The tagged scFvs toxintargeting agents and the anti-tag monoclonal antibodies were effectivefor treating subjects at risk for or having been contacted with adisease agent.

The use of small binding agents to direct the decoration of toxin withantibody permits new strategies for the development of agents withimproved therapeutic and commercial properties. Examples herein showthat a single recombinant heterodimeric binding protein/agent includingtwo or more high-affinity BoNT binding agents (camelid heavy-chain-onlyAb VH (VHH) domains) and two epitopic tags, co-administered with ananti-tag mAb, protected subjects from botulism caused negative symptomsand lethality. Further the binding protein resulted in antitoxinefficacy equivalent to and greater than conventional BoNT antitoxinserum in two different in vivo assays. Examples herein compareneutralizing or non-neutralizing binding agents administered with orwithout clearing antibody, and show the relative contributions of toxinneutralization and toxin clearance to antitoxin efficacy. Examplesherein show that both toxin neutralization and toxin clearancecontribute significantly to antitoxin efficacy in subjects. Toxinneutralization or toxin clearance using heterodimer binding proteinantitoxins sufficiently protected subjects from BoNT lethality in atherapeutically relevant, post-intoxication assay. Methods in Examplesherein optionally further include a clearing antibody for example amonoclonal anti-E-tag antibody.

It was observed in Examples herein that VHH binding agents thatneutralized toxin function significantly improved the antitoxin efficacyand even obviated the need for clearing antibody in a clinicallyrelevant post-intoxication BoNT/A assay. The methods, compositions andkits using the multimeric binding proteins described herein havewidespread application in antitoxin development and other therapies inwhich neutralization and/or accelerated clearance of a target moleculebenefits a patient. For example, the target molecule is an exogenousdisease agent that infects or is at risk to infect a patient. Exogenousdisease agent for example is a virus, a cancer cell, a fungus, abacterium, a parasite and a product thereof such as a pathogenicmolecule, a protein, a lipopolysaccharide, or a toxin. Alternatively,the molecule is an endogenous (body produced) molecule that is producedin the patient and that causes or produces harmful effects on thepatient. For example, the molecule is a hormone or a protein that isassociated with a disease or condition, e.g., inflammation, cancer,transplant rejection, kidney failure, or a defect in blood clotting suchas hemophilia and thrombophilia. In various embodiments, the diseaseagent is a toxin of C. difficile.

C. difficile is a gram-positive, spore forming, anaerobic bacterium thatis the leading cause of antibiotic-associated diarrhea, the severity ofwhich ranges from mild diarrhea to life threatening pseudomembranouscolitis (Bartlett J G. 2002 N Engl J Med 346:334-9 and Feng et al.PCT/US10/58701 filed Dec. 2, 2010, each of which is incorporated byreference in its entirety). Pathogenic C. difficile strains excreteexotoxins A (TcdA) and B (TcdB) that have been intimately linked to itspathogenicity. Both TcdA and TcdB are enterotoxic, capable of inducingintestinal epithelial damage and increasing mucosal permeability, andhence are thought to be responsible for the pathogenesis of C.difficile-associated colitis (Kelly C P et al. 1998 Annu Rev Med49:375-90). C. difficile has emerged as a leading cause ofhospital-acquired enteric infections with rapidly escalating annualhealth care costs in the United States (Kyne L et al. 2002 Clin InfectDis 34:346-353). The severity of C. difficile-associated infectionsranges from mild diarrhea to life threatening pseudomembranous colitis(Bartlett J G et al. 2002 N Engl J Med 346:334-339; Borriello S P 1998Antimicrob Chemother 41 Suppl C:13-19). Several hospital outbreaks of C.difficile-associated diarrhea (CDAD), with high morbidity and mortalityin the past few years in North America, have been attributed to thewidespread use of broad-spectrum antibiotics.

The emergence of more virulent C. difficile strains contributes also tothe increased incidence and severity of the disease (Loo V G et al. 2005N Engl J Med 353:2442-2449; McDonald L C et al. 2005 N Engl J Med353:2433-2441). Antibiotic usage results in a reduction of commensalmicroflora in the gut, which permits C. difficile to proliferate moreextensively, leading to the further production of toxins (Owens J R etal. 2008 Clinical Infectious Diseases 46(s1):S19-S31). C. difficileinfection (CDI) includes a range of symptoms varying from mild diarrheato severe fulminate lethal disease (Kuijper E J et al. 2007 Curr OpinInfect Dis 20(4):376-383). Recent outbreaks of highly virulent C.difficile strains (McDonald L C et al. 2005 N Engl J Med353(23):2433-2441; Loo V G et al. 2005 N Engl J Med 353(23):2442-2449)have increased the urgency to devote greater resources towards theunderstanding of the molecular, genetic, and biochemical basis for thepathogenesis, with a view to use such information to develop novelpreventive and treatment modalities.

A cell-based immunocytotoxicity assay for detecting C. difficile toxinsdescribed in Feng et al. (PCT/US2009/003055 published Nov. 19, 2009 asWO 2009/139919) uses an anti-C. difficile toxin A (TcdA) monoclonalantibody, named A1H3, which substantially enhanced the activity of TcdAon Fc gamma receptor I (FcγRI)-expressing cells (He X, Sun X, Wang J, etal. Antibody-enhanced, Fc{gamma}R-mediated endocytosis of C. difficiletoxin A. Infect Immun 2009). Feng et al. shows use of A1H3 enhancingantibody, in combination with an electronic sensing system to develop areal-time and ultrasensitive assay for the detection of biologicalactivity of C. difficile toxins.

Toxin A (TcdA) and toxin B (TcdB) are the major virulence factorscontributing to pathogenic C. difficile strains. These strains areenterotoxic, inducing intestinal epithelial cell damage, disruptingepithelium tight junctions leading to increased mucosal permeability(Pothoulakis C et al. 2001 Am J Physiol Gastrointest Liver Physiol280:G178-183; Riegler M et al. 1995 J Clin Invest 95:2004-2011; SavidgeT C et al. 2003 Gastroenterology 125:413-420). Moreover, these toxinsinduce production of immune mediators, leading to subsequent neutrophilinfiltration and severe colitis (Kelly C P et al. 1994 J Clin Invest93:1257-1265; Kelly C P et al. 1998 Annu Rev Med 49:375-390). TcdA andTcdB are structurally homologous, and contain a putative N-terminalglucosyltransferase and a cysteine proteinase domain, a transmembranedomain, and a C-terminal receptor binding domain (von Eichel-Streiber Cet al. 1996 Trends Microbiol 4:375-382) (Jank T et al. 2008 Trends inmicrobiology 16:222-229; Voth D E et al. 2005 Clin Microbiol Rev18:247-263).

Interaction between the toxin C-terminus and the host cell receptorsinitiates a receptor-mediated endocytosis (Florin I et al. 1983 BiochimBiophys Acta 763:383-392; Karlsson K A 1995 Curr Opin Struct Biot5:622-635; Tucker K D et al. 1991 Infect Immun 59:73-78). Although theintracellular mode of action remains unclear, it has been proposed thatthe toxins undergo conformational change at low pH in the endosomalcompartment, leading to membrane insertion and channel formation (FlorinI et al. 1986 Microb Pathog 1:373-385; Giesemann T et al. 2006 J BiolChem 281:10808-10815; Henriques B et al. 1987 Microb Pathog 2:455-463;QaDan M et al. 2000 Infect Immun 68:2470-2474). A host cofactor is thenrequired to trigger a second structural change which is accompanied byan immediate autocatalytic cleavage and release of theglucosyltransferase domain into cytosol (Pfeifer G et al. 2003 J BiolChem 278:44535-44541; Reineke J e al. 2007 Nature 446:415-419; Rupnik Met al. 2005 Microbiology 151:199-208). Once the glucosyltransferasedomain reaches the cytosol, it inactivates proteins of the Rho/Raefamily, leading to alterations of cytoskeleton and ultimately cell death(Just I et al. 1995 Nature 375:500-503; Sehr P et al. 1998 Biochemistry37:5296-5304).

The clinical manifestation of CDI is highly variable, from asymptomaticcarriage, to mild self-limiting diarrhea, to the more severepseudomembranous colitis. The prevalence of systemic complication anddeath in CDI has become increasingly common (Siemann M et al. 2000Intensive care medicine 26:416-421). In life-threatening cases of CDI,systemic complications are observed, including cardiopulmonary arrest(Johnson. S et al. 2001 Annals of internal medicine 135:434-438), acuterespiratory distress syndrome (Jacob S S et al. 2004 Heart Lung33:265-268), multiple organ failure (Dobson G et al. 2003 Intensive caremedicine 29:1030), renal failure (Cunney R J et al. 1998 Nephrol DialTransplant 13:2842-2846), and liver damage (Sakurai T et al. 2001 JInfect Dis 33:69-70). The exact reason for these negative complicationsis unclear, and may be caused by entry of the toxin into the circulationand systemic dissemination (Hamm E E et al. 2006 Proc Natl Acad Sci USA103:14176-14181).

Standard therapy depends on treatment with vancomycin or metronidazole,neither of which is fully effective (Zar et al. 2007 Clinical InfectiousDiseases 45:302-307). Moreover, an estimated 15% to 35% of thoseinfected with C. difficile relapse following treatment (Barbut et al.2000 J Clin Microbiol 38: 2386-2388; Tonna et al: Postgrad Med J 81:367). Unfortunately, the primary treatment option for recurrent CDI isstill metronidazole or vancomycin. Other options, such as probiotics,toxin-absorbing polymer and anion-exchange resins, have limited efficacy(Gerding, D. N., Muto, C. A. & Owens, R. C., Jr. 2008 Clin Infect Dis 46Suppl 1: S32-42). Therefore, immune-based therapies are the probably themost promising approaches to control the disease. Antibodies specificfor both of these toxins, and not against TcdA or TcdB alone, protectagainst toxigenic C. difficile infection in a hamster model (Libby etal, 1982 Infect Immun 36: 822-829; Fernie et al, 1983 Dev Biol Stand 53:325; and Kim et al, 2006 Infection and immunity 74: 6339). Human serumantibodies specific for both TcdA and TcdB are associated also withprotection against symptomatic disease and recurrence. Recent phase IIclinical trial led by Merck demonstrated that the systemicallyadministered human IgG monoclonal antibodies against TcdA and TcdBprevents disease relapse in CDI patients (Lowy et al, 2010 The NewEngland journal of medicine 362: 197). However, the treatment involvedthe injection of a large quantity of two individual antibodies againsteach toxin.

Examples herein show a new approach to the development of antitoxinsthat employs a single recombinant protein to promote toxin decorationwith multiple copies of a single monoclonal antibody leading to itsneutralization and clearance from the body. The methods, compositions,and kits herein are useful for treating a great number of the mostcommon pathogenic biological targets by accelerating neutralization andclearance from the subject or patient.

Examples herein show that camelid VHH binding domains, which havemultiple commercial advantages over scFvs due in part to the ease andreduced cost of producing VHHs, were effective as toxin targeting agentsboth with and without being administered with clearing antibody. Animportant advantage of VHHs is the ability of medical professionals andscientists to express these binding agents as heterodimers in which eachcomponent VHH remains fully functional. The multimeric fusion proteinscontaining at least two VHH binding regions resulted in the componentVHHs binding to different epitopes on the same toxin target. Withoutbeing limited by any particular theory or mechanism of action, it isbelieved that incorporation of two epitope tags on the heterodimersresulted in decoration of the toxin with two clearing antibodies at eachepitope, and resulted in a total of four monoclonal clearing antibodiesbinding to the heterodimers on the toxin. In addition, with certainheterodimers the decoration promoted efficient toxin clearance. Eitherneutralization or clearance or both are important mechanisms ofremediating toxin exposure. As each double-tagged heterodimeric bindingagent was bound only to only two monoclonal antibodies, theheterodimeric agent itself may not be effectively cleared by lowaffinity Fc receptors unless actually bound to the toxin.

The ability of antitoxin antibodies to protect mammalian subjects fromthe symptoms of toxin exposure is influenced by several factors that aredescribed herein. Examples herein used intoxication models and variedthe dose of antitoxin agent and the timing of antitoxin administrationrelative to exposure to toxin in order to determine whether both thedose and the timing of the antitoxin are factors that influenceantitoxin efficacy. In addition, examples herein analyzed the role thataffinity of the antibody for the toxin has on the ability of theantibody to bind (K_(on)) and remain bound (K_(off)) to the toxin andexert its effect. Data show that the ability of the antibodymonomer/heterodimer to inhibit the enzymatic activity of the toxinand/or prevent its entry into target cells (i.e. neutralization) is amajor factor in effective antitoxin treatment of subjects. Specificallydata show that the greater the binding affinity of the binding proteinto the target molecule, the greater the potential neutralization andclearance of the binding protein. Examples herein show also that themultimeric binding proteins promoted the clearance of the toxin from theserum and minimized further negative symptoms or lethality by the targetmolecule or disease agent. A portion of this work was published Jan. 6,2012 in the Public Library of Science One and was entitled, “A NovelStrategy for Development of Recombinant Antitoxin Therapeutics Tested ina Mouse Botulism”, authored by Jean Mukherjee, Jacqueline M. Tremblay,Clinton E. Leysath, Kwasi Ofori, Karen Baldwin, Xiaochuan Feng, DanielaBedenice, Robert P. Webb, Patrick M. Wright, Leonard A. Smith, SaulTzipori, and Charles B. Shoemaker (Mukherjee J. et al. 2012 PLoS One.7(1):e29941), which is incorporated by reference herein in its entirety.An aspect of the invention provides a method for treating a subject atrisk for exposure to or exposed to a disease agent, the methodincluding: contacting the subject with at least one recombinantheteromultimeric neutralizing binding protein including two or multiplebinding regions, such that the binding regions are not identical, andeach binding region specifically binds a non-overlapping portion of thedisease agent, such that the binding protein neutralizes the diseaseagent, thereby treating the subject for exposure to the disease agent.

In various embodiments of the method, the binding protein includes atleast one tag. For example the tag is a molecule or epitope that isattached or genetically fused to the binding protein and/or bindingregions. The tag in various embodiments of the method inducesendogeneous clearance of the disease agent from the body in vivo. Forexample the tag includes SEQ ID NO: 15. In a related embodiment, the tagincludes an antibody epitope.

In certain embodiments of the method, the binding protein is selectedfrom: a single-chain antibody (scFv); a recombinant camelidheavy-chain-only antibody (VHH); a shark heavy-chain-only antibody(VNAR); a microprotein; a darpin; an anticalin; an adnectin; an aptamer;a Fv; a Fab; a Fab′; and a F(ab′)₂. In an embodiment, the bindingprotein is heterodimeric, for example the binding protein has greaterpotency than each individual monomer. In alternative embodiments, theheteromultimeric neutralizing binding protein is multimeric and themultimeric components are associated non-covalently or covalently.

The binding protein in certain embodiments of the method includes alinker that separates multimeric components of the binding regions. Invarious embodiments, the linker includes at least one selected from: apeptide, a protein, a sugar, or a nucleotide. For example, the linkerincludes amino acid sequence GGGGS (SEQ ID NO: 54), or includes aminoacid sequence GGGGSGGGGSGGGGS (SEQ ID NO: 55) or a portion thereof. In arelated embodiment, the linker is a flexible linker located withinsubunits/domains of the binding protein, such that the linker does notnegatively affect the function of the binding protein to the diseaseagent. For example the linker includes amino acid sequences/residuesincluding serine and glycine, and in various embodiments is at leastabout three to five amino acids long, or about five to eight amino acidslong, or about eight to fifteen amino acids long.

In certain embodiments, the disease agent is a biological target orbiological molecule. For example, the biological target or thebiological molecule is naturally occurring within the subject, forexample a molecule or compound synthesized by the subject. An example ofa biological molecule synthesized by the subject is an IgE that isassociated with an allergy or an auto antibody or an MHC protein (e.g.,HLA class I antigens A and B and HLA class II antigen DR) associatedwith an autoimmune disease. For example the autoimmune disease isselected from: lupus erythematosus, Graves' disease, rheumatoidarthritis, Sjögren's syndrome, myasthenia gravis, and Hashimoto'sthyroiditis.

The disease agent in various embodiments of the method includes aplurality of non-identical disease agents, for example two or morebacterial toxins, or a viral toxin and a fungal species. In variousembodiments, the binding regions of the binding protein are specific toeach non-identical disease agent and bind to and neutralize theplurality of disease agents.

In various embodiments of the method, the disease agent is at least oneselected from: a virus, a cancer cell, a fungus, a bacterium, a parasiteand a product thereof such as a pathogenic molecule, a protein, alipopolysaccharide, and a toxin. In certain embodiments, the toxinincludes a protein, a lipid, a lipopolysaccharide, and a small moleculetoxin such as an aflatoxin or a dinoflagellate toxin. The toxin forexample is a Botulinum neurotoxin comprising a serotype selected from:A, B, C, D, E, F, and G. In certain embodiments of the method, the toxinis a Clostridium exotoxin comprising toxin A (TcdA) and toxin B (TcdB).

In various embodiments of the method, the toxin is at least one selectedfrom: staphylococcal α-hemolysin, staphylococcal leukocidin, aerolysincytotoxic enterotoxin, a cholera toxin, Bacillus cereus hemolysis IItoxin, a Helicobacter pylori vacuolating toxin, a Bacillus anthracistoxin, a cholera toxin, a Escherichia coli serotype O157:H7 toxin, aEscherichia coli serotype O104:H7 toxin, a lipopolysaccharide endotoxin,a Shiga toxin, a pertussis toxin, a Clostridium perfringens iota toxin,a Clostridium spiroforme toxin, a Clostridium difficile toxin A, aClostridium difficile toxin B, a Clostridium septicum α toxin, and aClostridium botulinum C2 toxin. In a related embodiment of the method,the disease agent is an infectious strain, for example a bacterialstrain or a viral strain. In a related embodiment, the disease agent isa Gram-negative strain or a Gram positive strain.

The bacterium in various embodiments of the method is selected from thegroup consisting of: B. anthracis, B. cereus, C. botulinum, C.difficile, C. perfringens, C. spiroforme, and V. cholerae.

In certain embodiments, the binding regions bind to different diseaseagents, such that the binding protein is specific for a plurality ofdisease agents, e.g., a Clostridium toxin and an Escherichia toxin. Forexample, the binding protein includes a chimeric fusion protein specificto at least two different disease agents described herein. In certainembodiments of the method, the binding protein is a humanized antibodyderived from a non-human species for example a mouse, a rabbit, analpaca, a llama, or horse.

In a related embodiment, the method further includes observingneutralizing of the disease agent by the binding protein and/or survivalof the subject. In certain embodiments of the method, observing furtherincludes measuring an amount of the disease agent or a disease agentproduct in a sample from the subject. In various embodiments, the sampleis selected from: a cell, a fluid, and a tissue. For example, the fluidis at least one selected from: blood, serum, plasma, mucosal fluid,saliva, cerebrospinal fluid, semen, tears, and urine. In certainembodiments of the method, the cell or the tissue is at least oneselected from: fecal; vascular; epithelial; endothelial; dermal; dental;connective; muscular; neuronal; facial; cranial; soft tissue includingcartilage and collagen; brain; bone; bone marrow; joint tissue; andarticular joints. For example, the method includes collecting the fluid,the cell, or the tissue from a biopsy. In certain embodiments, themethod includes collecting the fluid, the cell, or the tissue from an exvivo sample or aliquot. Alternatively, the method includes collectingfrom fluid, cell, or tissue that is in vivo or in situ.

The method further includes in a related embodiment observing areduction or a remediation in at least one pathology symptom associatedwith the disease agent. In various embodiments, the method furtherincludes prior to contacting the subject with the binding protein,observing and/or detecting in the subject an indicium of the exposure tothe disease agent selected from: diarrhea, vomiting, breathingdifficulty, fever, inflammation, bleeding, pain, numbness, loss ofconsciousness, tissue necrosis, or organ failure. For example, thesubject is a transplant recipient or an immunosuppressed patient.

In a related embodiment, the method further includes contacting thesubject with the binding protein at a period of time such as seconds,minutes, or hours after observing the indicium. Alternatively, themethod further includes contacting the subject with the binding proteinseconds, minutes, hours, or days prior to an event that is associatedwith the risk for the exposure. For example, the method includescontacting the subject prior to or after the subject's entering apotentially hazardous or dangerous environment such as biohazardfacility, a combat zone, or a hazardous waste site.

The method in related embodiments includes contacting the subject withthe binding protein by injecting a solution including the bindingprotein into the subject. In various embodiments, injecting involves atleast one selected from: subcutaneous, intravenous, intramuscular,intraperitoneal, intradermal, intramedullary, transcutaneous, andintravitreal. In various embodiments of the method, contacting thesubject with binding protein includes at least one technique selectedfrom: topically, ocularly, nasally, bucally, orally, rectally,parenterally, intracisternally, intravaginally, or intraperitoneally. Ina related embodiment, contacting the subject involves using anapplicator, for example the applicator is a syringe, a needle, asprayer, a sponge, a gel, a strip, a tape, a bandage, a tray, a string,or a device used to apply a solution to a cell or a tissue.

In a related embodiment of the method, contacting the subject with thebinding protein includes administering to the subject a source ofexpression of the binding protein. In various embodiments of the method,the source of expression of the binding protein is a nucleotide sequenceencoding the binding protein, such that the source of the expressionincludes at least one selected from the group consisting of: a nakednucleic acid vector, bacterial vector, and a viral vector. For example,the bacterial vector is derived from at least one selected from thegroup consisting of: E. coli, Bacillus spp, Clostridium spp,Lactobacillus spp, and Lactococcus spp.

In a related embodiment of the method, contacting further includesadministering the vector, for example the naked nucleic acid vector, thebacterial vector, or the viral vector.

In a related embodiment, the nucleotide acid sequence further includesan operably linked signal for promoting expression of the bindingprotein. For example, the signal includes a mammalian promoter or anon-viral promoter. In a related embodiment, the method involvesengineering the binding protein or the source of expression of thebinding protein (e.g., viral vector or bacterial vector) using adimerizer sequence for example having an amino acid sequence includingSEQ ID NO: 94 or a portion or homolog thereof. For example, thedimerizer sequences forms a covalent bond or disulfide linkage betweenat least two amino acid sequences to form a homodimer, a heterodimer, ora multimer. The method in various embodiments includes, prior tocontacting, engineering the binding protein using an agent thatmultimerizes at least one binding region or a multimer, e.g., aheterodimer, a heterotrimer, and a heterotetramer, to form the bindingprotein.

In a related embodiment of the method, the viral vector is derived fromat least one selected from: an adenovirus, an adeno-associated virus, aherpesvirus, and a lentivirus. The method in various embodiments furtherincludes contacting the subject with a gene delivery vehicle selectedfrom at least one of: a liposome, a lipid/polycation (LPD), a peptide, ananoparticle, a gold particle, and a polymer. For example, the genedelivery vehicle specifically targets a cell or tissue in the body bycontacting or binding a receptor located on the cell or tissue.

An aspect of the invention provides a pharmaceutical composition fortreating a subject at risk for exposure to or exposed to a diseaseagent, the pharmaceutical composition including: at least onerecombinant heteromultimeric neutralizing binding protein including twoor more binding regions, such that the binding regions are notidentical, and each binding region specifically binds a non-overlappingportion of the disease agent, such that the binding protein neutralizesthe disease agent, thereby treating the subject for exposure to thedisease agent.

In a related embodiment, the composition is compounded with apharmaceutically acceptable buffer or diluent. For example thecomposition is compounded for parenteral administration such asintravenous, mucosal administration, topical administration, or oraladministration.

In various embodiments, the subject is at least one selected from: ahuman, a dog, a cat, a goat, a cow, a pig, and a horse. For example, thehuman subject is a: sick child or adult, health-care profession (e.g.,doctor and nurse), aid worker, member of the military, or animmunosuppressed patient such as a transplant recipient. In certainembodiments, the pharmaceutical composition is formulated to protect thesubject against the exposure, for example that exposure includes apicogram amount, nanogram amount, microgram amount, or gram amount ofthe disease agent or a plurality of disease agents.

The binding protein or binding regions in various embodiments of thecomposition is selected from the group of a single-chain antibody(scFv); a recombinant camelid heavy-chain-only antibody (VHH); a sharkheavy-chain-only antibody (VNAR); a microprotein; a darpin; ananticalin; an adnectin; an aptamer; a Fv; a Fab; a Fab′; and a F(ab′)₂.In various embodiments, the binding regions are of a different type, forexample at least one binding region is a VHH and at least other bindingregion is a scFv, an Fab or any of the types described herein.

The composition in various embodiments further includes at least oneagent selected from the group of: an antitoxin, an anti-inflammatory, ananti-tumor, an antiviral, an antibacterial, an anti-mycobacterial, ananti-fungal, an anti-proliferative, an anti-apoptotic, an anti-allergy,and an anti-immune suppressant.

In an embodiment, the composition further includes a labeled detectablemarker selected from the group consisting of: detectable, fluorescent,colorimetric, enzymatic, radioactive, and the like. For example, themarker is detectable in a sample taken from the subject, the sampleexemplified by a cell, a fluid or a tissue. In a related embodiment, themarker includes a peptide, a protein, a carbohydrate, and a polymer.

In an embodiment of the composition, the binding protein includes alinker that separates the binding regions. The linker in a relatedembodiment separates the binding regions and/or subunits of themultimeric protein. In certain embodiments, the binding protein includesa linker that covalently joins each binding region of the heterodimericor the multimeric protein. In various embodiments, the linker includesat least one selected from the group of a peptide, a protein, a sugar,or a nucleic acid. In a related embodiment, the linker includes aminoacid sequence GGGGS (SEQ ID NO: 54) or a portion thereof. In a relatedembodiment, the linker includes amino acid sequence GGGGSGGGGSGGGGS (SEQID NO: 55) or a portion thereof or multiples thereof. The linker invarious embodiments stabilizes the binding protein and does not preventthe respective binding of the binding regions to the disease agent or toa plurality of disease agents.

In various embodiments of the pharmaceutical composition, the bindingprotein and/or binding regions include at least one tag that is attachedor genetically fused to the binding protein and/or binding regions. Thetag for example is a peptide, sugar, or DNA molecule that does notinhibit or prevent binding of the binding protein and/or binding regionsto the disease agent. In various embodiments, the tag is at least about:three to five amino acids long, five to eight amino acids long, eight totwelve amino acids long, twelve to fifteen amino acids long, or fifteento twenty amino acids long. For example, the tag includes SEQ ID NO: 15.

In various embodiments, the disease agent for which the binding proteinis specific is at least one selected from: a virus, a cancer cell, afungus, a bacterium, a parasite and a product thereof such as apathogenic molecule, a protein, a lipopolysaccharide, or a toxin. Inrelated embodiments of the composition, the toxin includes a protein, alipid, a lipopolysaccharide, and a small molecule toxin such as anaflatoxin or a dinoflagellate toxin. For example, the toxin is aBotulinum neurotoxin comprising a serotype selected from: A, B, C, D, E,F, and G. In various embodiments of the composition, the toxin is atleast one selected from: staphylococcal α-hemolysin, staphylococcalleukocidin, aerolysin cytotoxic enterotoxin, a cholera toxin, Bacilluscereus hemolysis II toxin, a Helicobacter pylori vacuolating toxin, aBacillus anthracis toxin, a cholera toxin, a Escherichia coli serotypeO157:H7 toxin, a Escherichia coli serotype O104:H7 toxin, alipopolysaccharide endotoxin, a Shiga toxin, a pertussis toxin, aClostridium perfringens iota toxin, a Clostridium spiroforme toxin, aClostridium difficile toxin A, a Clostridium difficile toxin B, aClostridium septicum a toxin, and a Clostridium botulinum C2 toxin. Incertain embodiments, the disease agent includes a plurality ofnon-identical disease agents such that the binding regions of thebinding protein bind to and neutralize the plurality of disease agents.

In various embodiments of the composition, the bacterium for which thebinding protein is specific is selected from: B. anthracis, B. cereus,C. botulinum, C. difficile, C. perfringens, V. cholerae, and C.spiroforme. In a related embodiment, the bacterium is a virulentbacterium or apathogenic bacterium.

The composition in various embodiments is compounded or formulated for aroute of delivery selected from the group of: topical, ocular, nasal,bucal, oral, rectal, parenteral, intracisternal, invaginal, andintraperitoneal.

In various embodiments of the composition, the binding protein isspecific for a toxin which is a C. botulinum toxin, and the bindingregions of the binding protein includes a recombinant camelidheavy-chain-only antibody, and the composition includes an amino acidsequence selected from the group:

(VHH H7, SEQ ID NO: 56)LVQVGGSLRLSCVVSGSDISGIAMGWYRQAPGKRREMVADIFSGGSTDYAGSVKGRFTISRDNAKKTSYLQMNNVKPEDTGVYYCRLYGSGDYWGQGTQVTVSSAHHSEDP;(VHH B5, SEQ ID NO: 57)LVHPGGSLRLSCAPSASLPSTPENPFNNMVGWYRQAPGKQREMVASIGLRINYADSVKGRFTISRDNAKNTVDLQMDSLRPEDSATYYCHIEYTHYWGKGTLVTVSSEPKTPKPQ; and(H7/B5 heterodimer, SEQ ID NO: 58)QVQLVESGGGLVQVGGSLRLSCVVSGSDISGIAMGWYRQAPGKRREMVADIFSGGSTDYAGSVKGRFTISRDNAKKTSYLQMNNVKPEDTGVYYCRLYGSGDYWGQGTQVTVSSAHHSEDPTSAIAGGGGSGGGGSGGGGSLQGQLQLVESGGGLVHPGGSLRLSCAPSASLPSTPFNPFNNMVGWYRQAPGKQREMVASIGLRINYADSVKGRFTISRDNAKNTVDLQMDSLRPEDSATYYCHIEYTHYWGKGTLVTVSSEPKTPKPQ.

In a related embodiment of the composition, the binding protein isspecific for a toxin which is a C. difficile toxin A, and the bindingregion of the binding protein includes a recombinant camelidheavy-chain-only antibody having an amino acid sequence selected fromthe group of:

(AH3, SEQ ID NO: 59)QVQLVETGGLVQPGGSLRLSCAASGFTLDYSSIGWFRQAPGKEREGVSCISSSGDSTKYADSVKGRFTTSRDNAKNTVYLQMNSLKPDDTAVYYCAAFRATMCGVFPLSPYGKDDWGKGTLVTVSSEPKTPKPQP; (AA6, SEQ ID NO: 60)QLQLVETGGGLVQPGGSLRLSCAASGFTESDYVMTWVRQAPGKGPEWIATINTDGSTMRDDSTKGRFTISRDNAKNTLYLQMTSLKPEDTALYYCARGRVISASAIRGAVRGPGTQVTVSSEPKTPKPQP; (A3H, SEQ ID NO: 61)QVQLVESGGGLVQPGGSLRLSCAASGFTLDYYAIGWERQAPGKEREGVSGISSVDGSTYYADSVRGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAADQSPIPIHYSRTYSGPYGMDYWGKGTLVTVSSAHHSEDP; (AC1, SEQ ID NO: 62)QLQLVESGGGLVQPGGSLRLSCAASGFTLDYYAIGWERQAPGKEREGVSGISFVDGSTYYADSVKGRFAISRGNAKNTVYLQMNSLKPEDTAVYYCAADQSSIPMHYSSTYSGPSGMDYWGKGTLVTVSSEPKTPKPQP; (A11G, SEQ ID NO: 63)QLQLVETGGGLVQAGGSLRLSCAASGRTLSNYPMGWFRQAPGKEREFVAAIRRIADGTYYADSVKGRFTISRDNAWNTLYLQMNGLKPEDTAVYFCATGPGAFPGMVVTNPSAYPYWGQGTQVTVSSEPKTPKPQP; (AE1, SEQ ID NO: 64)QLQLVESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVSGISSSDGSTYYADSVKGRFTISRDNATNTVYLQMNSLKPEDTAVYYCAADQAAIPMHYSASYSGPRGMDYWGKGTLVTVSSEPKTPKPQP; (SEQ ID NO: 87)MSDKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPCKMIAPILDEIADEYQGKLTVAKLNIDQNPGTAPKYGIRGIPTLLLFKNGEVAATKVGALSKGQLKEFLDANLAGSGSGHMHHHHHHSSGLVPRGSGMKETAAAKFERQHMDSPDLGTDDDDKAMAISDPNSQVQLVESGGGLVQPGGSLRLSCEASGFTLDYYGIGWFRQPPGKEREAVSYISASARTILYADSVKGRFTISRDNAKNAVYLQMNSLKREDTAVYYCARRRFSASSVNRWLADDYDVWGRGTQVAVSSEPKTPKPQTSAIAGGGGSGGGGSGGGGSLQAMAAASQVQLVESGGGLVQTGGSLRLSCASSGSIAGFETVTWSRQAPGKSLQWVASMTKTNNEIYSDSVKGRFIISRDNAKNTVYLQMNSLKPEDTGVYFCKGPELRGQGIQVTVSSEPKTPKPQPARR; and, (SEQ ID NO: 95)MSDKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPCKMIAPILDEIADEYQGKLTVAKLNIDQNPGTAPKYGIRGIPTLLLFKNGEVAATKVGALSKGQLKEFLDANLAGSGSGHMHHHHHHSSGLVPRGSGMKETAAAKFERQHMDSPDLGTDDDDKAMAISDPNSQVQLVETGGLVQPGGSLRLSCAASGFTLDYSSIGWFRQAPGKEREGVSCISSSGDSTKYADSVKGRETTSRDNAKNTVYLQMNSLKPDDTAVYYCAAFRATMCGVFPLSPYGKDDWGKGTLVTVSSEPKTPKPQPTSAIAGGGGSGGGGSGGGGSLQAMAAAQLQLVETGGGLVQPGGSLRLSCAASGFTFSDYVMTWVRQAPGKGPEWIATINTDGSTMRDDSTKGRFTISRDNAKNTLYLQMTSLKPEDTALYYCARGRVISASAIRGAVRGPGTQVTVSSEPKTPKPQPARQTSPSTVRLESRVRELEDRLEELRDELERAERRANEMSIQLDEC.

In certain embodiments of the composition, the binding protein isspecific for a toxin which is a C. difficile toxin B, and the bindingregion of the binding protein includes a recombinant camelidheavy-chain-only antibody having an amino acid sequence selected fromthe group consisting of:

(2D, SEQ ID NO: 65)QVQLVESGGGLVQPGGSLRLSCAASGFSLDYYGIGWFRQAPGKERQEVSYISASAKTKLYSDSVKGRFTISRDNAKNAVYLEMNSLKREDTAVYYCARRRFDASASNRWLAADYDYWGQGTQVTVSSEPKTPKPQ; (20s, SEQ ID NO: 66)QVQLVESGGGLVQAGGSLRLSCVSSERNPGINAMGWYRQAPGSQRKLVAIWQTGGSLNYADSVKGRFTTSRDNLKNTVYLQMNSLKPUDTAVYYCYLKKWRDQYWGQGTQVTVSSEP KTPKPQ;(5D, SEQ ID NO: 67)QVQLVESGGGLVQPGGSLRLSCEASGFTLDYYGIGWFRQPPGKERHAVSYISASARTILYADSVKGRFTISRDNAKNAVYLQMNSLKREDTAVYYCARRRFSASSVNRWLADDYDVWGRGTQVAVSSEPKTPKPQ; (E3, SEQ ID NO: 68)OVQLVESGGGLVQTGGSLRLSCASSGSIAGFETVTWSROAPGKSLQWVASMTKINNEIYSDSVKGRFIISRDNAKNTVYLQMNSLKPEDTGVYFCKGPELRGQGIQVTVSSEPKTPKPQ;(7F, SEQ ID NO: 69)QVQLVESGGGLVEAGGSLRLSCVVTGSSFSTSTMAWYRQPPGKQREWVASFTSGGAIKYTDSVKGRFTMSRDNAKKMTYLQMENLKPEDTAVYYCALHNAVSGSSWGRGTQVTVSSE PKTPKPQ;(5E, SEQ ID NO: 70)VQLVESGGGLVQAGGSLRLSCAASGLMFGAMTMGWYRQAPGKEREMVAYITAGGTESYSESVKGRFTISRINANNMVYLQMTNLKVEDTAVYYCNAHNFWRTSRNWGQGTQVTVS SEPKTPKP;(B12, SEQ ID NO: 71)VQLVESGGGLVQAGDSLTLSCAASESTFNTFSMAWFRQAPGKEREYVAAFSRSGGTTNYADSVKGRATISTDNAKNTVYLHMNSLKPEDTAVYFCAADRPAGRAYFQSRSYNYWGQGTQVTVSSAHHSEDP; (A11, SEQ ID NO: 72)VQLVESGGGSVQIGGSLRLSCVASGFTFSKNIMSWARQAPGKGLEWVSTISIGGAATSYADSVKGRFTISRDNANDTLYLQMNNLKPEDTAVYYCSRGPRTYINTASRGQGTQVTVSSEP KTPKP;(AB8, SEQ ID NO: 73)VQLVESGGGLVQAGGSLRLSCVGSGRNPGINAMGWYRQAPCiSQRELVAVWQTGGSTNYADSVKGRFTISRDNLKNTVYLQMNSLKPEDTAVYYCYLKKWRDEYWGQGTQVTVSSAH HSEDP;(C6, SEQ ID NO: 74)VQLVESGGGLVQAGESLRLSCVVSESIFRINTMGWYRQTPGKQREVVARITLRNSTTYADSVKGRFTISRDDAKNTLYLKMDSLKPEDTAVYYCHRYPMFRNSPYWGQGTQVTVSSEPK TPKP;(C12, SEQ ID NO: 75)VQLVESGGGLVQAGESLRLSCVVSESIFRINTMGWYRQTPGKQREVVARITLRNSTTYADSVKGRFTISRDDAKNTITIXMDSIXPEDTAVYYCHRYPLIFRNSPYWGQGTQVTVSSEPK TP;(A1, SEQ ID NO: 76); SEQ ID NO: 87; and SEQ ID NO: 95VQLVESGGGLVQAGGSLRLSCAAPGLTFTSYRMGWFRQAPGKEREYVAAITGAGATNYADSAKGRFTISKNNTASTVHLQMNSLKPEDTAVYYCAASNRAGGYWRASQYDYWGQGTQVTVSSAHHSEDP.

In related embodiments of the composition, the binding protein isspecific for a toxin which is a Shiga toxin, and the binding region ofthe binding protein includes a recombinant camelid heavy-chain-onlyantibody having an amino acid sequence selected from the group:

(JET-A9, SEQ ID NO: 77)QVQLVETGGGLAQAGDSLRLSCVEPGRTLDMYAMGWIRQAPGEEREFVASISGVGGSPRYADSVKGRFTISKDNTKSTIWLQMNSLKPEDTAVYYCAAGGDIYYGGSPQWRGQGTRVT VSSEPKTPKPQ;(JGG-D4, SEQ ID NO: 78)QVQLVESGGGLVQAGGSLRLSCAASGRINGDYAMGWFRQAPGEEREFVAVNSWIGGSTYYTDSVKGRFTLSRDNAKNTLSLQMNSLKPEDTAVYYCAAGHYTDFPTYFKEYDYWGQGTQVTVSSEPKTPKPQ; (JEN-D10, SEQ ID NO: 79)QVQLVETGGLVQAGGSLRLSCAASGVPFSDYTMAWFRQAPGKEREVVARITWRGGGPYYGNSGNGRFAISRDIAKSMVYLHMDSLKPEDTAVYYCAASRLRPALASMASDYDYWGQGTQVSVSSEPKTPKPQ; (JGH-G1, SEQ ID NO: 80)QVQLVESGGGLVQPGESLRLSCVASASTFSTSLMGWVRQAPGKGLESVAEVRTTGGTFYAKSVAGRFTISRDNAKNTLYLQMNSLKAEDTGVYYCTAGAGPIATRYRGQGTQVTVSSA HHSEDP;(JEU-A6, SEQ ID NO: 81)QVQLVESGGGLVQPGGSLKLSCAASGFTLADYVTVWFRQAPGKSREGVSCISSSRGTPNYADSVKGRATVSRNNANNTVYLQMNGLKPDDTAIYYCAAIRPARLRAYRECLSSQAEYDYWGQGTQVTVSSAHHSEDP; (JEU-D2, SEQ ID NO: 82)QVQLVESGGGLVQPGGSLGLSCAMSGTTQDYSAVGWFRQAPGKEREGVSCISRSGRRTNYADSVRGRFTISRDNAKDTVYLQMNSLKPDDTAVYYCAARKTDMSDPYYVGCNGMDYWGKGTLVTVSSAHHSEDP; (JGH-G9, SEQ ID NO: 83)QVQLVESGGGLVQPGGSLTLSCTASGFTLNSYKIGWFRQAPGKEREGVSCINSGGNLRSVEGRFTISRDNTKNTVSLHMDSLKPEDTGVYHCAAAPALNVFSPCVLAPRYDYWGQGTQV TVSSAHHSEDP;(JFD-A4, SEQ ID NO: 84)QVQLVESGGGLVQPGGSLRLSCAASGFTLGSYHIGWFRHPPGKEREGTSCLSSRGDYTKYAEAVKGRFTISRDNTKSTVYLQMNNLKPEDTGIYVCAAIRPVLSDSHCTLAARYNYWGQGTQVTVSSAHHSEDP; (JFD-A5, SEQ ID NO: 85)QVQLVESGGGLVQPGGSLRLSCAALEFTLEDYAIAWFRQAPGKEREGVSCISKSGVTKYTDSVKGRFIVARDNAKSTVILQMNNLRPEDTAVYNCAAVRPVFVDSVCTLATRYTYWGEGTQVTVSSAHHSEDP; and (JGG-G6, SEQ ID NO: 86)QVQLVETGGGLVQPGGSLKLSCAASEFTLDDYHIGWFRQAPGKEREGVSCINKRGDYINYKDSVKGRFTISRDGAKSTVFLQMNNLRPEDTAVYYCAAVNPVFPDSRCTLATRYTHWGQGTQVTVSSAHHSEDP.

In various embodiments, the amino acid sequence of the compositionfurther includes an amino acid analog, an amino acid derivative, or aconservative substitution of an amino acid residue. The binding proteinin various embodiments includes an amino acid sequence that issubstantially identical to the amino acid sequence of SEQ ID NOs: 56-87and 95. In related embodiments, substantially identical means that theamino acid sequence of the binding protein has at least about 50%identity, at least about 60% identity, at least about 65% identity, atleast about 70% identity, at least about 75% identity, at least about80% identity, at least about 85% identity, at least about 90% identity,at least about 95% identity, at least about 97% identity, at least about98% identity, or at least about 99% identity to the amino acid sequenceof SEQ ID NOs: 56-87 and 95. Alternatively, the binding protein isencoded by at least one nucleotide sequence or the protein includesamino acid sequence selected from the group of SEQ ID NOs: 1-87 and 95,and substantially identical to any of these sequences.

The composition in various embodiments further includes the bindingprotein or a source of expression of the binding protein selected fromthe group of: a purified binding protein preparation; a nucleic acidvector with a gene encoding the binding protein; a viral vector encodingthe binding protein; and a naked nucleic acid encoding the bindingprotein which is expressed from the DNA. In related embodiments, theviral vector is derived from a genetically engineered genome of at leastone virus selected from: an adenovirus, an adeno-associated virus, aherpes virus, and a lentivirus.

In a related embodiment of the composition, the binding protein isheterodimeric. In various embodiments, the heterodimeric binding proteinincludes a first binding region and a second binding region. For examplethe first binding region and the second binding region include VHHs, andthe first binding region binds specifically to a C. difficile TcdA andthe second binding region binds specifically to a C. difficile TcdB.

An aspect of the invention provides a kit for treating a subject exposedto or at risk for exposure to a disease agent including: apharmaceutical composition for treating a subject at risk for exposureto or exposed to a disease agent, the pharmaceutical compositionincluding: at least one recombinant heteromultimeric neutralizingbinding protein comprising a plurality binding regions, such that thebinding regions are not identical, and each binding region specificallybinds a non-overlapping portion of the disease agent, such that thebinding protein neutralizes the disease agent, thereby treating thesubject for exposure to the disease agent; a container; and,instructions for use. In various embodiments, the instructions for useinclude instructions for a method for treating a subject at risk forexposure to or exposed to a disease agent using the pharmaceuticalcomposition.

In various embodiments of the kit, the binding protein is selected fromthe group of: a single-chain antibody (scFv); a recombinant camelidheavy-chain-only antibody (VHH); a shark heavy-chain-only antibody(VNAR); a microprotein; a darpin; an anticalin; an adnectin; an aptamer;a Fv; a Fab; a Fab′; and a F(ab′)₂.

In a related embodiment of the kit, the binding protein includes alinker. In various embodiments, the linker includes at least oneselected from: a peptide, a protein, a sugar, or a nucleic acid. Forexample, the linker includes amino acid sequence GGGGS (SEQ ID NO: 54),or GGGGSGGGGSGGGGS (SEQ ID NO: 55), or a portion thereof. Alternatively,the linker includes a single amino acid or a plurality of amino acids.

In related embodiments of the kit, the disease agent for which thebinding protein and binding regions are specific is selected from: avirus, a cancer cell, a fungus, a bacterium, a parasite, and a productof one of those such as a pathogenic molecule, a protein, alipopolysaccharide, or a toxin. In related embodiments, the toxin forwhich the binding protein is specific is a Botulinum neurotoxinincluding a serotype selected from: A, B, C, D, E, F, and G. In variousembodiments of the kit, the toxin for which the binding protein isspecific is at least one selected from the group of: staphylococcalα-hemolysin, staphylococcal leukocidin, aerolysin cytotoxic enterotoxin,a cholera toxin, a Bacillus cereus hemolysis II toxin, a Helicobacterpylori vacuolating toxin, a Bacillus anthracisi toxin, a cholera toxin,an Escherichia coli serotype O157:H7 toxin, an Escherichia coli serotypeO104:H7 toxin, a lipopolysaccharide endotoxin, a Shiga toxin, apertussis toxin, a Clostridium perfringens iota toxin, a Clostridiumspiroforme toxin, a Clostridium difficile toxin A, a Clostridiumdifficile toxin B, a Clostridium septicum a toxin, and a Clostridiumbotulinum C2 toxin. In certain embodiments, the binding regions of thebinding protein are specific to different classes of disease agents,e.g., each of the plurality of binding regions is different and isspecific for an agent from bacteria, virus, fungus, cancer, and apathogenic molecule. For example a binding region is specific for avirus and another binding region is specific for a bacterium.

In a related embodiment of the kit, the binding protein is specific fora toxin which is a C. botulinum toxin, and the binding region includes arecombinant camelid heavy-chain-only antibody, such that thepharmaceutical composition includes the binding protein that has anamino acid sequence selected from the group consisting of: SEQ ID NO:56, SEQ ID NO: 57, SEQ ID NO: 58, or a portion thereof.

In a related embodiment of the kit, the binding region of the bindingprotein is specific for a toxin which is a C. botulinum toxin A, suchthat the binding region of the binding protein includes a recombinantcamelid heavy-chain-only antibody having an amino acid sequence selectedfrom the group of: SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ IDNO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 87, SEQ ID NO: 95, anda portion thereof.

In a related embodiment of the kit, the toxin for which the bindingprotein is specific is a C. difficile toxin B, and the binding region ofthe binding protein includes a recombinant camelid heavy-chain-onlyantibody having an amino acid sequence selected from: SEQ ID NO: 65, SEQID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70,SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO:75, SEQ ID NO: 76, SEQ ID NO: 87, SEQ ID NO: 95, and a portion thereof.In certain embodiments, the binding protein and/or binding regions areencoded by a nucleotide sequence or the binding protein and/or regionsinclude an amino acid sequence selected from the group of SEQ ID NOs:1-87 and 95, or are substantially identical to these sequences.

In a related embodiment, the binding protein is specific for a Shigatoxin, and the binding region of the binding protein includes arecombinant camelid heavy-chain-only antibody having an amino acidsequence selected from: SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84,SEQ ID NO: 85, and SEQ ID NO: 86.

An aspect of the invention provides a composition including at least oneamino acid sequence selected from the group of: SEQ ID NO: 59, SEQ IDNO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69,SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO:74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ IDNO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 94,SEQ ID NO: 95 or a portion thereof. The composition in variousembodiments includes an amino acid sequence that is substantiallyidentical to the amino acid sequence of SEQ ID NOs: 59-86. In relatedembodiments, substantially identical means an amino acid sequence thathas at least 60% identity, at least 65% identity, at least 70% identity,at least 75% identity, at least 80% identity, at least 85% identity, atleast 90% identity, at least 95% identity, at least about 97% identity,at least about 98% identity, or at least 99% identity to an amino acidsequence of any of SEQ ID NOs: 56-87 and 95.

An aspect of the invention provides a method for treating a subject atrisk for exposure to or exposed to a plurality of disease agents, themethod including: contacting the subject with at least one recombinantheteromultimeric neutralizing binding protein including two or morebinding regions, such that the binding protein neutralizes at least two(plurality) of disease agents, thereby treating the subject for exposureto the plurality of disease agents.

In a related embodiment of the method, the at least two of the bindingregions are identical. Alternatively, the at least two binding regionsinclude at least two non-identical binding regions. In relatedembodiments of the method, the binding protein is at least one selectedfrom the group of: a heterodimer, a trimer, a tetramer, a pentamer, anda hexamer. In various embodiments, the tetramer includes a homodimer ofa heterodimer, for example a heterodimer of AH3 and AA6 as is shown inSEQ ID NO: 95.

In various embodiments, the plurality from which the exemplary diseaseagents are selected from a virus, a cancer cell, a fungus, a bacterium,a parasite and a product thereof such as a pathogenic molecule, aprotein, a lipopolysaccharide, or a toxin. For example the diseaseagents include toxins such as TcdA and TcdB.

In related embodiments of the method, the binding protein includes atleast one selected from the group of SEQ ID NOs: 56-87 and 95 or aportion or a homologue.

In related embodiments of the method, the binding protein is selectedfrom the group of: a single-chain antibody (scFv); a recombinant camelidheavy-chain-only antibody (VHH); a shark heavy-chain-only antibody(VNAR); a microprotein; a darpin; an anticalin; an adnectin; an aptamer;a Fv; a Fab; a Fab′; and a F(ab′)₂. In a related embodiment of themethod, the binding protein includes a linker located between each ofthe multimeric components of the binding regions. In variousembodiments, the linker is at least one selected from the group of: apeptide, a protein, a sugar, or a nucleic acid. For example, the linkercomprises amino acid sequence GGGGS (SEQ ID NO: 54) or amino acidsequence GGGGSGGGGSGGGGS (SEQ ID NO: 55).

In a related embodiment, the method further includes prior tocontacting, engineering the binding protein using a dimerization agent.In a related embodiment, the dimerization agent includes amino acidsequence TSPSTVRLESRVRELEDRLEELRDELERAERRANEMSIQLDEC (SEQ ID NO:94), ora portion thereof.

In various embodiments of the method, the plurality of disease agents isat least two selected from the group of: Staphylococcal α-hemolysin,Staphylococcal leukocidin, aerolysin cytotoxic enterotoxin, a choleratoxin, Bacillus cereus hemolysis II, and Helicobacter pylori vacuolatingtoxin, Bacillus anthracis, cholera toxin, Escherichia coli serotypeO157:H7, Escherichia coli serotype O104:H7, lipopolysaccharideendotoxin, Shiga toxin, pertussis toxin, Clostridium perfringens iotatoxin, Clostridium spiroforme toxin, Clostridium difficile toxin A,Clostridium difficile toxin B, Clostridium septicum a toxin, andClostridium botulinum C2 toxin. In related embodiments of the method,the binding protein includes at least one selected from the group of:SEQ ID NOs: 56-87 and 95.

Binding Agent

The binding agent or binding protein is in one embodiment, a moleculethat binds to a portion of a target molecule, disease agent, or diseaseagent target. The binding protein treats the subject by any or all ofseveral mechanisms, including promoting clearance, phagocytosis,neutralization, inhibition, and activation of the immune response. Theterm “binding agent” or “binding protein”, includes in addition tofull-length antibodies, molecules such as antibody fragments (e.g.,single chain antibodies, and VHHs), microproteins (also referred to ascysteine knot proteins or knottins), darpins, anticalins, adnectins,peptide mimetic molecules, aptamers, synthetic molecules, and refers toany composition that binds to a target and/or disease agent and elicitsan immune effector activity against the molecule target and/or diseaseagent. In certain embodiments, the binding protein is a recombinantmultimeric neutralizing binding protein including two or more bindingregions, such that the binding regions are not identical, and eachand/or disease agent. Alternatively, the binding protein includesbinding regions that bind specifically to different types of diseaseagents such as different types of pathogenic molecules such as bacteria,viruses, fungi, allergens, and toxins. For example at least one bindingregion of the binding protein bind to a virus surface protein, and atleast one different binding regions binds to a bacterial toxin.

The multimeric neutralizing binding protein herein in certainembodiments includes one or a plurality of epitopic tags. In certainembodiments, the binding protein includes a linker that covalentlyconnects each binding region of the heterodimer. For example, the linkeris a single amino acid or a sequence of a plurality of amino acids thatdoes not affect or reduce the stability, orientation, binding,neutralization, and/or clearance characteristics of the binding regionsand binding protein. In certain embodiments, each binding region isspecific to a non-identical disease agent. For example the bindingprotein in certain embodiments includes a binding region specific to abacterium or bacterial toxin, and at least one other binding region isspecific to a virus, fungus, allergen, or to a non-identical bacteriumor bacterial toxin. For example, a multimeric binding protein in certainembodiments has binding regions specific to a TcdA and to a TcdA or to aShiga toxin, or the respective binding regions are specific to each of aBotulinum toxin and a virus.

In certain embodiments, the binding protein neutralizes or inhibits themolecule target and/or disease agent for example by preventing thedisease agent entry into cells. In certain embodiments, the bindingprotein upon being administered to the subject neutralizes the toxinand/or triggers an antibody mediated effector activity in the subject.

The binding protein is in certain embodiments a monomer (e.g., a singleunit), or includes a covalently bound protein including a plurality ofmonomers such as for example a dimer, a trimer, a tetramer, a pentamer,an octamer, a 10-mer, a 15-mer, a 20-mer, or any multimer. In certainembodiments, the binding protein is a monomer and the binding proteinhas one binding region that binds to an epitope of the molecule targetand/or disease agent. Alternatively, the binding protein in certainembodiments has two or more connected or joined monomers each with abinding region and each binding to an epitope of a disease agent or to aplurality of epitopes of disease agents. The multimeric binding proteinin certain embodiments includes the same monomer. Alternatively themultimeric binding protein includes monomers or binding regions or acombination thereof (i.e., heteromulteric). Accordingly, the multimerscan be homogeneous such that each includes two or more monomers having abinding region that binds to the same site of a disease agent.Alternatively the multimers are heterogeneous and include two or moremonomers having a binding region that binds to two or more differentsites of one or more disease agents. The heterogeneous multimers(heteromultimers) bind non-overlapping portions of the molecule targetand/or disease agent. In various embodiments, the binding protein is ahomodimer of a heterodimer or a heterotrimer. In a related embodiment,the heteromultimers bind a plurality of non-identical epitopes on aplurality of disease agents.

In certain embodiments the binding protein includes a single tag,multiple tags, for example each multimeric binding protein includes twoor more tags on each component binding region (i.e., monomer).Alternatively, the heterodimer comprises no tag attached to the monomersand/or linker. In certain embodiments, presence of the tag on oroperably fused to the binding protein and/or binding regionsynergistically induces clearance of the disease agent from the body.For example the tag attached to the binding protein induces an immuneresponse from a patient or subject contacted with a pharmaceuticalcomposition containing the tagged-binding protein. In certainembodiments the tag includes a portion (e.g., conserved, unique,in-activated, and non-functional) of a pathogenic molecule. In certainembodiments, the tag is an adjuvant. See Gerber et al. U.S. Pat. No.7,879,333 issued Feb. 1, 2011 which is incorporated by reference hereinin its entirety. For example, the tag is a peptide, carbohydrate,polymer, or nucleic acid that is effective for enhancing neutralizationand/or clearance of the disease agent or plurality of disease agents.

The multimeric binding protein in certain embodiments is a heterodimerhaving two tags, one tag attached to each monomer, or alternatively theheterodimer includes one tag on each monomer or one tag total on one ofthe two monomers. The term “heterodimer” includes a single proteinhaving two different monomers are joined by a linker. Data herein shownthat a heterodimers having two E-tags effectively protected animalsexposed to hundreds-fold and/or thousands-fold the lethal dose of asingle disease agent such as a C. difficile toxin A. Examples hereinshow that recombinant multimeric binding proteins, having two or morenon-identical binding regions, administered to subjects either before orafter contact with a disease agent resulted in comparable and betterantitoxin efficacy than serum-based polyclonal antitoxins.

The binding agents/proteins described herein include bindingagent/protein portions, regions, and fragments. For example, the bindingprotein is an antibody and, in certain embodiments the binding proteinincludes antibody fragments. The term “antibody fragment” refers toportion of an immunoglobulin having specificity to an molecule targetand/or disease agent, or a molecule involved in the interaction orbinding of the molecule target and/or disease agent. The term “antibodyfragment” encompasses fragments from binding protein, for example bothpolyclonal and monoclonal antibodies including transgenically producedantibodies, single-chain antibodies (scFvs), recombinant Fabs, andrecombinant heavy-chain-only antibodies (VHHs), e.g., from any organismproducing VHH antibody such as a camelid, a shark, or a designed VHH.

VHHs are antibody-derived therapeutic proteins that contain the uniquestructural and functional properties of naturally-occurring heavy-chainantibodies. VHH technology is based on fully functional antibodies fromcamelids that lack light chains. These heavy-chain antibodies contain asingle variable domain (VHH) and two constant domains (CH2 and CH3). Thecloned and isolated VHH domain is a stable polypeptide harboring theantigen-binding capacity of the original heavy-chain antibody. SeeCastorman et al. U.S. Pat. No. 5,840,526 issued Nov. 24, 1998; andCastorman et al. U.S. Pat. No. 6,015,695 issued Jan. 18, 2000, each ofwhich is incorporated by reference herein in its entirety. VHHs arecommercially available from Ablynx Inc. (Ghent, Belgium) under thetrademark of Nanobodies™.

Suitable methods of producing or isolating antibody fragments having therequisite binding specificity and affinity are described herein andinclude for example, methods which select recombinant antibody from alibrary, by PCR (See Ladner U.S. Pat. No. 5,455,030 issued Oct. 3, 1995and Devy et al. U.S. Pat. No. 7,745,587 issued Jun. 29, 2010, each ofwhich is incorporated by reference herein in its entirety).

Functional fragments of antibodies, including fragments of chimeric,humanized, primatized, veneered or single chain antibodies, can also beproduced. Functional fragments or portions of the foregoing antibodiesinclude those which are reactive with the disease agent. For example,antibody fragments capable of binding to the disease agent or portionthereof, including, but not limited to scFvs, Fabs, VHHs, Fv, Fab, Fab′and F(ab′)₂ are encompassed by the invention. Such fragments can beproduced by enzymatic cleavage or by recombinant techniques. Forinstance, papain or pepsin cleavage are used generate Fab or F(ab′)₂fragments, respectively. Antibody fragments are produced in a variety oftruncated forms using antibody genes in which one or more stop codonshas been introduced upstream of the natural stop site. For example, achimeric gene encoding a F(ab′)₂ heavy chain peptide portion can bedesigned to include DNA sequences encoding the CH₁ peptide domain andhinge region of the heavy chain. Accordingly, the present inventionencompasses a polynucleic acid that encodes the binding proteindescribed herein (e.g., a binding fragment with a tag). Binding proteinsin certain embodiments are made as part of a multimeric protein, themonomer or single binding region (e.g., antibody fragments,microproteins, darpins, anticalins, adnectins, peptide mimeticmolecules, aptamers, synthetic molecules, etc) can be linked. Anycombination of binding protein or binding region types can be linked. Inan embodiment, the monomer or binding region of a multimeric bindingprotein can be linked covalently. In another embodiment, a monomerbinding protein can be modified, for example, by attachment (directly orindirectly (e.g., via a linker or spacer)) to another monomer bindingprotein. A monomer in various embodiments is attached or geneticallyfused to another monomer e.g., by recombinant protein that is engineeredto contain extra amino acid sequences that constitute the monomers.Thus, the DNA encoding one monomer is joined (in reading frame) with theDNA encoding the second monomer, and so on. Additional amino acids incertain embodiments are encoded between the monomers that produce anunstructured region separating the different monomers to better promotethe independent folding of each monomer into its active conformation orshape. Commercially available techniques for fusing proteins are used invarious embodiments to join the monomers into a multimeric bindingprotein of the present invention.

The term “antagonist” as used herein includes proteins or polypeptidesthat bind to the disease agent, inhibit function of the disease agent,and are included in certain embodiments to the binding region of thebinding protein.

A binding protein includes any amino acid sequence that binds to thedisease agent or target including molecules that have scaffolds.Examples of binding proteins having scaffolds are DARPins, Anticalins,and AdNectins. DARPins are derived from natural ankyrin repeat proteinsand bind to proteins including e.g., human receptors, cytokines,kinases, human proteases, viruses and membrane proteins (MolecularPartners AG Zurich Switzerland). Anticalins are derived from lipocalins,and comprise a hypervariable loops supported by a conserved β-sheetframework, which acts as a binding protein. (Pieris AG, Germany). Thescaffold for anticalins are lipocalins. AdNectins are derived from humanfibronectin (e.g., the scaffold), and bind to targets of various medicalconditions and are commercially available from Adnexus (Waltham, Mass.).See also Alexandru et al. U.S. Pat. No. 7,867,724 issued Jan. 11, 2011,which is incorporated by reference herein in its entirety. In certainembodiments, the binding protein having the scaffold is encoded by anucleotide sequence or the binding protein includes an amino acidsequence that is substantially identical or homologous to the sequencesdescribed herein, for example SEQ ID NO: 1-87 and 95. Recombinantmultimeric binding proteins herein include amino acid sequences from abinding protein sequence having conservative sequence modifications. Asused herein, the term “conservative sequence modifications” refers toamino acid modifications that do not significantly affect or alter thecharacteristics (e.g., neutralization, clearance, binding, stability,and orientation) of the binding protein, i.e., amino acid sequences ofbinding protein that present these side chains at the same relativepositions will function in a manner similar to the binding protein. Suchconservative modifications include amino acid substitutions, additionsand deletions. Modification of the amino acid sequence of recombinantmultimeric binding protein is achieved using any known technique in theart e.g., site-directed mutagenesis or PCR based mutagenesis. Suchtechniques are described in Sambrook et al., Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Press, Plainview, N.Y., 1989 andAusubel et al., Current Protocols in Molecular Biology, John Wiley &Sons, New York, N.Y., 1989. Conservative amino acid substitutions aremodifications in which the amino acid residue is replaced with an aminoacid residue having a similar side chain such as replacing a small aminoacid with a different small amino acid, a hydrophilic amino acid with adifferent hydrophilic amino acid, etc.

Examples herein show that a molecule target and/or disease agent isbound by a binding protein, the molecule target and/or disease agentexemplified by a bacterial toxin released by the pathogen, for example abotulinum toxin. Botulinum toxin serotypes A to G are synthesized byorganisms including Clostridium botulinum, Clostridium baratii, andClostridium butyricum. Simpson, L. L 2004 Annu. Rev. Pharmacol. Toxicol.44: 167-193. C. botulinum produces serotypes A to G, C. baratii producesserotype F, and C. butyricum produces serotype E only. The structuresand substrates for each of the botulism toxin serotypes as well as theserotype specific cleavage sites have been determined, and the mechanismof toxin killing has been elucidated. The botulinum toxin actspreferentially on peripheral cholinergic nerve endings to blockacetylcholine release, and causes disease (i.e., botulism) and can beused to treat disease (e.g., dystonia). Ibid., Abstract. Thetoxigenicity of botulinum toxin depends on penetration of the toxinthrough cellular and intracellular membranes. Thus, toxin that isingested or inhaled binds to epithelial cells and is transported to thegeneral vascular circulation. Toxin that reaches peripheral nerveendings binds to the cell surface then penetrates the plasma membrane byreceptor-mediated endocytosis and the endosome membrane by pH-inducedtranslocation. Ibid., Abstract. Internalized toxin acts in the cytosolas a metalloendoprotease to cleave polypeptides that are essential forexocytosis.

Examples herein show binding proteins/agents that specifically bind eachof a variety of distinct serotypes of a microbial neurotoxin that causesbotulism, BoNT/A and BoNT/B. The amino acid sequence of the bindingagents include scFvs and VHHs for example SEQ ID NOs: 2, 4, 6, 8, 10,12, 14, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50,52 or combinations or portions thereof. The corresponding nucleic acidsequences of binding agents are shown in SEQ ID NOs: 1, 3, 5, 7, 9, 11,13, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51or a combination thereof. In various embodiments the amino acid sequenceof the binding agents includes VHHs for example SEQ ID NO: 56-87 or 95or combinations or portions thereof. In certain embodiments, the bindingagent includes a tag that was engineered as a portion of the bindingagent, for example the tag has amino acid sequence of SEQ ID NO: 15, andis genetically fused to the carboxyl end of the binding agents. Incertain embodiments, the tag enhances ability of the binding protein toneutralize and/or clear the disease agent from the subject. FIG. 5 showsa phylogenetic tree of JDQ-B5 (SEQ ID NO: 24), a VHH binding agent thatspecifically binds to BoNT/A and other VHHs that compete with JDQ-B5 forbinding to BoNT/A. The length of the branches in the tree represents therelatedness of the sequences with the shorter branches indicatinggreater relatedness (i.e., homology) and the longer branches indicatingless homology of the amino acid sequences.

The present invention provides a number of different binding proteins,each having binding regions with specificity and affinity to targetdifferent areas of one or more disease agents. In an embodiment, two orthree binding proteins specific to different epitopes of a disease agentare used. In a disease having a number of disease agents involved incausing the disease or condition, such as botulism, multiple diseaseagents are targeted by the compositions and methods herein. In the caseof botulism, since any one of at least seven neurotoxin serotypes areinvolved, a pool/mixture of binding proteins is prepared containingbinding proteins for a plurality of known serotypes that cause thedisease in humans. Botulism is often caused by exposure to a single BoNTserotype, and it is generally difficult to quickly determine whichserotype is the cause. Thus, the standard of care in treating botulismincludes administration of a number of antibodies to protect againstmost if not all of the serotypes that cause the disease in human. Hence,it is appropriate to protect subjects from botulism, to stockpilebinding proteins that bind to several or preferably all known serotypesthat cause botulism.

The present invention in various embodiments further encompassescompositions that are multimeric binding proteins having two or moremonomers in which a monomer is exemplified by a VHH amino sequenceherein. In various embodiments, the composition includes at least oneselected from the group of SEQ ID NOs: 56-87 and 95. Compositionsfurther include nucleic acid sequences that encode the amino acidssequences herein, for example SEQ ID NO: 56-87 and 95. In certainembodiments, the monomer or binding region includes at least onesequence described herein, for example SEQ ID NOs: 1-87 and 95. Anembodiment of a multimeric binding protein includes two or more of theVHH sequences herein expressed as a single protein. Any combination oftwo or more of the VHH sequences forms a multimeric binding protein ofthe present invention. In a particular embodiment, the present inventionrelates to a heterodimer, i.e., protein, in which any two different VHHsequences herein are expressed as a single protein, i.e., linked andexpressed as a genetic fusion.

The binding protein in certain embodiments is a multimeric fusionprotein engineered and produced using a multimerization agent to form acomplex that effectively binds to and neutralizes a disease agent orplurality of disease agents. In certain embodiments, the multimerizationagent includes a dimerization sequence for example including an aminoacid sequence shown in SEQ ID NO: 94. For example the dimerzation agentcomplexes peptide fragments each containing at least: about five to 25amino acids, about 25 to 50 amino acids, about 50 to 100 amino acids,about 100 to 150 amino acids, and about 150 amino acids to about 200amino acids. Multimerization agents and methods of using the agents forforming multimeric binding proteins are shown herein in Example 21. Seealso Moore et al. U.S. Pat. No. 7,763,445 issued Jul. 24, 2012 andCarter et al. U.S. Pat. No. 8,216,865 issued Jul. 10, 2012, each ofwhich is incorporated by reference herein in its entirety.

The disease agent target is any from different classes of pathogens,infectious agents or other unwanted material. A multi-target approach iswithin the scope of the methods and compositions herein, exemplified bya binding protein that binds to a viral disease agent, a bacterialdisease agent, a parasite disease agent, a cancer cell, and a proteinproduced therefrom and any combination thereof. In various embodiments,a binding protein neutralizes a plurality of pathogens or unwantedmaterial. Examples herein show a VHH heterodimer that binds to andneutralizes both TcdA and TcdB.

The disease agent, pathogen or infectious agent that is neutralized bythe binding agent is any molecule, virus or bacterium that infects amammal (e.g., human, horse, dog, goat, and cow) or a mammalian cell. Incertain embodiments, the disease agent is a bacterium selected fromActinobacillus, Bacillus, Borrelia, Brucella, Campylobacter, Chlamydia,Clostridium, Coxiella, Enterococcus, Escherichia, Francisella,Hemophilus, Legionella, Mycobacterium, Neisseria, Pasteurella,Pneumophila, Pseudomonas, Rickettsia, Salmonella, Shigella,Staphylococcus, Streptococcus, Treponema, and Yersinia. Alternatively,the disease agent is a virus including for example humanimmunodeficiency virus, foot-and-mouth disease virus, avian influenzavirus, and sheep pox virus.

The binding agent in various embodiments binds to and neutralizes aninfectious agent and/or a disease agent associated with a pathologyresulting from overexpression of a self protein in the subject such asan immunoglobulin, a leukocyte, a cytokine, and a growth factor. Forexample the overexpression is of an inflammatory agent such as a tumornecrosis factor (e.g., TnFα) or an interleukin (IL) such as IL-1 beta,or IL-6. Alternatively, an infectious agent and/or a disease agent isassociated with expression of a mutated or modified molecule such as aprotein, a sugar, a glycoprotein, or expression of a cell carrying anucleotide sequence encoding the disease agent.

The binding agent in various embodiments binds to a cancer cell and/orcancer marker. For example the cancer cell includes a melanoma; acarcinoma (e.g., colon carcinoma); a pancreatic cancer; a sarcoma; alymphoma; a leukemia; a brain tumor such as glioma; a lung cancer; anesophageal cancer; a mammary (breast) cancer; a bladder cancer; aprostate cancer; a head and neck cancer; an ovarian cancer; a kidneycancer; or a liver cancer.

The binding agents described herein are used in certain embodiments totreat symptoms of an autoimmune disease, a class of disorder whichincludes Hashimoto's thyroiditis; idiopathic myxedema, a severehypothyroidism; multiple sclerosis, a demyelinating disease marked bypatches or hardened tissue in the brain or the spinal cord; myastheniagravis which is a disease having progressive weakness of muscles causedby autoimmune attack on acetylcholine receptors at neuromuscularjunctions; Guillain-Barre syndrome, a polyneuritis; systemic lupuserythematosis; uveitis; autoimmune oophoritis; chronic immunethrombocytopenic purpura; colitis; diabetes; Grave's disease, which is aform of hypothyroidism; psoriasis; pemphigus vulgaris; and rheumatoidarthritis (RA).

Molecule Target and Disease Agent Target

A molecule target and/or disease agent target is any target which isbiological (e.g., protein, sugar, carbohydrate, DNA, RNA) or chemical towhich the binding protein binds, and is any target associated with adisease, defect or negative condition. The molecule target or diseaseagent target is any molecule capable of being bound, or whose activityis altered (e.g., neutralized, reduced or ceased), or that can berecognized by immune effectors and leads for example to clearance,opsonization, killing, and phagocytosis. For example, the disease agenttarget in certain embodiments is a portion of a pathogen or a moleculereleased or secreted by the pathogen (e.g. toxin). A pathogen is anagent that causes a disease or condition, and includes a virus, cancercell, bacterium, parasite or pathogenic protein. The disease agenttarget includes a pathogenic protein that is derived from normal cells,such as prions. The pathogenic protein or other molecule that is diseaseagent target is either independent of the pathogen or is associated withor produced by the pathogen.

A virus is a microscopic particle that infects the cells of a biologicalorganism and replicates in the host cell. In various embodiments, viralantigens including viral proteins, are targeted by the binding protein.Binding proteins bind to molecules or receptors on the virus, and areneutralized and/or cleared using the methods described herein. Examplesof viruses that are neutralized and/or cleared by the binding proteinherein include Influenza, Rhinovirus, Rubeola, Rubella, Herpes,Smallpox, Chickenpox, Human Papilloma, Rabies, and HumanImmunodeficiency viruses.

A parasite is an organism that lives on or in a different organism.Parasites have or express molecules that are used as a target by thebinding agent. Types of parasites include endoparasites (e.g., parasitesthat live inside the body of the host) and ectoparasites (e.g.,parasites that live on the outside of the host's body). Examples ofparasites that are treated by the methods, compositions, and kits hereinare shown in Horvitz et al. U.S. patent publication 20110010782published Jan. 13, 2011. Exemplary parasites include a protozoan (e.g.,a plasmodium, a cryptosporidium, a microsporidium, and isospora), atick, a louse and a parasitic worm.

Molecules on cancer cells also are targets of the binding agent. Inrelated embodiments, the target is a protein on the cancer cell such asa cancer marker. Examples of proteins or receptors associated withcancer cells include CD33, HER2/neu, CA 125 (MUC16), prostate-specificantigen (PSA), and CD44.

The disease agent target in certain embodiments includes bacteriaincluding Gram negative and Gram positive bacteria. Examples ofpathogenic bacteria bound by the binding protein include Clostridium,Staphylococcus, Neisseria, Streptococcus, Moraxella, Listeria, any ofthe Enterobacteriaceae, Escherichia coli, Corynebacterium, Klebsiella,Salmonella, Shigella, Proteus, Pseudomonas, Haemophilus, Bordetella,Legionella, Campylobacter, Helicobacter, and Bacteroides.

Enterohemorrhagic Escherichia coli (EHEC) is an emerging food- andwater-borne pathogen that colonizes the distal ileum and colon andproduces potent cytotoxins (Donnenberg, “Infections due to Escherichiacoli and other enteric gram-negative bacilli,” in ACP Medicine, WebMDProfessional Publishing, Danbury Conn., Chapter 7, pp. 8-1 to 8-18,2005). After ingestion of contaminated food, humans develop symptomsranging from mild diarrhea to the severe, and at times life-threatening,hemolytic uremic syndrome (HUS). Currently, EHEC is the most commoncause of pediatric renal failure in the United States (Mead et al, EmergInfect Dis, 5:607-625, 1999). Several EHEC serotypes cause disease, butthe 0157 serotype is by far the most common cause of EHEC-relateddisease in North America, Europe and Japan (Feng, “Escherichia coli” inGarcia (ed.) Guide to Foodborne Pathogens. John Wiley and Sons, Inc.,pp. 143-162, 2001). See also Waldor et al., U.S. patent publicationnumber 2010/0092511 A1 published Apr. 15, 2012, which is incorporated byreference herein in its entirety.

Shiga toxins are a family of related toxins with two major groups, Stx1and Stx2 (Friedman et al., 2001 Curr Opin Microbiol 4 (2): 201-7). Thetoxins are named for Kiyoshi Shiga, who first described the bacterialorigin of dysentery caused by Shigella dysenteriae. The most commonsources for Shiga toxin are the bacteria S. dysenteriae and theShigatoxigenic group of Escherichia coli (STEC), which includesserotypes O157:H7, O104:114, and other enterohemorrhagic E. coli, EHEC(Spears et al. 2006 FEMS Microbiology Letter 187-202; Sandvig et al.2000 EMBO J. 19 (22): 5943-5950; and Krautz-Peterson et al. 2008Infection and Immunity 76(5) 1931-1939; and Vermeij U.S. Pat. No.7,807,184 issued Oct. 5, 2010, each of which is incorporated byreference herein in its entirety. Symptoms associated with Shigatoxin-exposure caused infection by EHEC include watery stool followed bysevere abdominal pain and bloody stool. Exposed persons developcomplications leading to HUS, encephalopathy, and even death (Masuda etal., U.S. Pat. No. 7,345,161 issued Mar. 18, 2008).

Methods for ascertaining the target molecule or disease agent aredescribed herein and depend on the type of molecule being inhibited. Forexample, in a case in which a class or group of bacteria are to beinhibited, conserved regions of bacteria are targeted, and bindingagents that bind to these targets are constructed. Methods for targetinga conserved region or polymorphic region of a nucleotide sequence thatencodes the target molecule, or the target molecule having an amino acidsequence are shown in Cicciarelli et al., U.S. patent publication number2005/0287129 A1 published Dec. 29, 2005 which is incorporated byreference herein in its entirety. In other embodiments, if a specificdisease agent such as a bacterium is to be inhibited, a non-conservedregion of the disease agent is targeted with the binding agents. Thebinding of the agents are determined and/or measured for example usingstandard assays, for example an enzyme-linked immunosorbent assay(ELISA), western blot and radioimmunoassay.

A molecule target or a disease agent target includes pathogenicmolecules including polypeptides or toxins to which the binding proteindescribed herein binds, neutralizes and/or clears. The term “pathogenicprotein” refers to a protein that can cause, directly or indirectly, adisease, or condition in an individual. A pathogenic protein is forexample a protein or a toxin produced by a bacterium, a virus, or acancer cell. A recombinant multimeric binding protein described hereinbinds non-overlapping areas of the disease agent target (e.g., a toxinproduced by a bacterium) and protects the subject from the pathology ofthe disease agent target by neutralizing and/or clearing the target. Thebinding protein protects subjects from negative symptoms caused byexposure to the disease agent target, and the risk of negative symptomscaused by a potential exposure to the target.

Anti-tag antibody described herein is used in various embodiments toeffect or facilitate effector functions. The anti-tag antibody includesfor example an immunoglobulin such as IgA, IgD, IgE, IgG, and IgM, andsubtypes thereof. In addition to monoclonal antibodies, polyclonalantibodies specific to the tag are used in the methods, compositions andkits described herein. Effector functions are performed for exampleimmune molecules interaction with the Fc portion of the immunoglobulin.Depending on the type of immunoglobulin chosen, the effector functionsresults in clearance of the disease agent (e.g., excretion, degradation,lysis or phagocytosis).

Mammalian antibody types IgA, IgD, IgE, IgG, and IgM, and antibodysubtypes are classified according to differences in their heavy chainconstant domains. Each immunoglobulin class differs in its biologicalproperties and characteristics. IgA is found for example in areascontaining mucus (e.g. in the gut, respiratory tract, and urogenitaltract) and prevents the colonization of mucosal areas by pathogens. IgDfunctions as a disease agent receptor on B cells.

IgE binds to allergens and triggers histamine release from mast cellsand also provides protection against helminths (worms). IgG, in fourforms, provides the majority of antibody-based immunity against invadingpathogens. IgM has a very high affinity for eliminating pathogens in theearly stages of B cell mediated immunity, and is expressed on thesurface of B cells and also in a secreted form.

Leukocytes such as mast cells and phagocytes have specific receptors onthe cell surface for binding antibodies. These Fc receptors interactwith the Fc region of classes of antibodies (e.g. IgA, IgG, IgE). Theengagement of a particular antibody with the Fc receptor on a particularcell triggers the effector function of that cell. For example,phagocytes function to perform phagocytosis, and mast cells function todegranulate. Effector functions generally result in destruction of aninvading microbe. In various embodiments, the type of immunoglobulin ischosen specifically for a type of desired effector function.

The present invention includes methods of administering one or morerecombinant multimeric binding proteins to a subject (e.g., human, cow,horse, pig, mouse, dog, and cat). The binding protein is administered incertain embodiments as a monomer, or as a multimeric binding proteincomprising a plurality of monomers having different binding regions. Themethods and compositions herein involve administration of one or moremultimeric binding agents that include monomers that each has a bindingregion that is specific to the disease agent. The binding agent forexample includes one or more tags. The binding agent/protein binds tothe target region on the disease protein. Administration of two or morebinding proteins (e.g., monomer binding proteins or multimeric bindingproteins), in various embodiments, increased the effectiveness of theantibody therapy, and reduced the severity of one or more negativesymptoms of exposure of the disease protein target. The binding proteinis administered in various embodiments as a single monomer, a mixture ofmultiple (e.g., two or more) monomers, a multimeric binding proteinincluding a plurality of monomers that are same or different, a mixtureof multiple (e.g., two or more) multimeric binding proteins comprisingmore than one monomer, or any combination thereof. Examples herein showthat administration of a binding protein containing more than one copyof the tag resulted in increased protection against a disease agenttarget, e.g., botulinum toxin serotype A. A single anti-tag antibodytype in certain embodiments binds to all binding proteins having a tag.In certain embodiments in which the binding proteins have multiplecopies (e.g., two or more) of the same tag, the anti-tag antibody bindsto each copy of the tag on the binding protein. The phrase, “antibodytherapeutic proteins” or “antibody therapeutic preparation” refers toone or more compositions that include at least one binding protein andoptionally at least one anti-tag antibody. The multimeric bindingprotein preparation in certain embodiments contains additional elementsincluding carriers as described herein.

The administration of the one or more binding proteins and/or anti-tagantibody is performed in related embodiments simultaneously orsequentially in time. The binding protein in certain embodiments isadministered before, after or at the same time as another bindingprotein or the anti-tag antibody, providing that the binding proteinsand/or the anti-tag antibodies are administered close enough in time tohave the desired effect (e.g., before the binding proteins have beencleared by the body). Thus, the term “co-administration” is used hereinto mean that the binding proteins and another binding protein or theanti-tag antibody are administered at time points to achieve effectivetreatment of the disease, and reduction in the level of the pathogen(e.g., virus, bacteria, cancer cell, proteins associated therewith, orcombination thereof) and symptoms associated with it. The methods of thepresent invention are not limited by the amount of time in between whichthe binding proteins and/or anti-tag antibody are administered;providing that the compositions are administered close enough in time toproduce the desired effect. In certain embodiment, the binding proteinsis administered only, alternatively the binding protein and/or anti-tagantibody are premixed and administered together. The binding proteinsand/or anti-tag antibody are in certain embodiments co-administered withother medications or compositions suitable to treating the diseaseagent.

The binding protein in certain embodiments is administered prior to thepotential risk of exposure to the disease target agent to protect thesubjects from symptoms of the disease agent target. For example, thebinding protein and/or clearing antibody is administered minutes, hoursor days prior to the risk of exposure. Alternatively, the bindingprotein is administered contemporaneously to the risk of exposure to thedisease agent target, or slightly after the risk of exposure. Forexample, the binding protein is administered to a subject at the momentthe subjects contacts, enters or passes through an environment (e.g.,room, hallway, building, and field) containing the risk of exposure tothe disease agent.

The methods of the present invention include treating a bacterialdisease, a parasitic infection, a viral disease, a cancer, smallunwanted molecule, a protein or a toxin associated therewith. This isaccomplished by administering the binding proteins and anti-tagantibodies described herein to the affected individual or individual atrisk. Administration ameliorates or reduces the severity of one or morethe symptoms of the disease or condition. The presence, absence orseverity of symptoms is measured for example using tests and diagnosticprocedures known in the art. Presence, absence and/or level of thedisease agent are measured in certain embodiments using methods known inthe art. Symptoms or levels of the disease agent can be measured at oneor more time points (e.g., before, during and after treatment, or anycombination thereof) during the course of treatment to determine if thetreatment is effective. A decrease or no change in the level of thedisease agent, or severity of symptoms associated therewith indicatesthat treatment is working, and an increase in the level of the diseaseagent, or severity of symptoms indicates that treatment is not working.Symptoms and levels of disease agents are measured in variousembodiments using methods known in the art. Symptoms that are monitoredin certain embodiments include fever, plain including headache, jointpain, muscular pain, difficulty breathing, lethargy, and impairedmobility, appetite and unresponsiveness. Toxin protection is assessed asincreased survival and reduction or prevention of symptoms. Methods,compositions and kits using the binding protein decrease and alleviatethe symptoms of the disease target agent and also improve survival fromexposure to the agent.

The antibody therapeutic agents including one or more binding proteinsor agents, and/or an anti-tag antibody are administered in variousembodiments with one or more pharmaceutical carriers. The teems“pharmaceutically acceptable carrier” and a “carrier” refer to anygenerally acceptable excipient or drug delivery device that isrelatively inert and non-toxic. The binding agents and anti-tag antibodyare administered with or without a carrier. Exemplary carriers includecalcium carbonate, sucrose, dextrose, mannose, albumin, starch,cellulose, silica gel, polyethylene glycol (PEG), dried skim milk, riceflour, magnesium stearate, and the like. Suitable formulations andadditional carriers are described in Remington's PharmaceuticalSciences, (17th Ed., Mack Pub. Co., Easton, Pa.), the teachings of whichare incorporated herein by reference in their entirety. The bindingagents and anti-tag antibody are administered systemically or locally(e.g., by injection or diffusion).

Suitable carriers (e.g., pharmaceutical carriers) include, but are notlimited to sterile water, salt solutions (such as Ringer's solution),alcohols, polyethylene glycols, gelatin, carbohydrates such as lactose,amylose or starch, magnesium stearate, talc, silicic acid, viscousparaffin, fatty acid esters, hydroxymethylcellulose, polyvinylpyrolidone, etc. The binding protein preparations are sterilized and, ifdesired, mixed with auxiliary agents, e.g., lubricants, preservatives,stabilizers, wetting agents, emulsifiers, salts for influencing osmoticpressure, buffers, coloring, and/or aromatic substances and the likewhich do not deleteriously react with the active compounds. The bindingprotein preparations in certain embodiments are combined where desiredwith other active substances, e.g., enzyme inhibitors, to reducemetabolic degradation. A carrier (e.g., a pharmaceutically acceptablecarrier) is used optionally in certain embodiments to administer one ormore binding agents and an anti-tag antibody.

The binding agents and anti-tag antibodies in certain embodiments areadministered topically (as by powders, ointments, or drops), orally,rectally, mucosally, sublingually, parenterally, intracisternally,intravaginally, intraperitoneally, bucally, ocularly, or intranasally,depending on preventive or therapeutic objectives and the severity andnature of a exposure or risk of exposure to the disease agent target.The composition in various embodiments is administered in a single doseor in more than one dose over a period of time to confer the desiredeffect.

An effective amount of compositions of the present invention variesaccording to choice of the binding agent, the particular compositionformulated, the mode of administration and the age, weight and conditionof the patient, for example. As used herein, an effective amount of thebinding agents and/or anti-tag antibody is an amount which is capable ofreducing one or more symptoms of the disease or conditions caused by themolecule target or disease agent target. Dosages for a particularpatient are determined by one of ordinary skill in the art usingconventional considerations, (e.g. by means of an appropriate,conventional pharmacological protocol).

A composition in certain embodiments includes one or more nucleotidesequences described herein that encode the binding protein. In variousembodiments, a nucleotide sequence is either present as a mixture or inthe form of a DNA molecule a multimer. A various embodiments, thecomposition includes a plurality of nucleotide sequences each encodingthe binding protein including a monomer or polypeptide, or anycombination of molecules described herein, such that the binding proteinis generated in situ. In such compositions, a nucleotide sequence isadministered using any of a variety of delivery systems known to thoseof ordinary skill in the art, including nucleic acid expression systems,bacterial and viral expression systems. Appropriate nucleic acidexpression systems contain appropriate nucleotide sequences operablylinked for expression in the patient (such as a suitable promoter andterminating signal). Bacterial delivery systems involve administrationof a bacterium (such as Bacillus-Calmette-Guerrin) that expresses thepolypeptide on its cell surface. In an embodiment, the DNA can beintroduced using a viral expression system (e.g., vaccinia or other poxvirus, retrovirus, or adenovirus), which uses a non-pathogenic(defective), replication competent virus. Techniques for incorporatingDNA into such expression systems are well known to those of ordinaryskill in the art. The DNA can also be “naked,” as described, forexample, in Ulmer et al., Science 259:1745-1749, 1993 and reviewed byCohen, Science 259:1691-1692, 1993. The uptake of naked DNA can beincreased by coating the DNA onto biodegradable beads, which areefficiently transported into recipient cells.

Systems or kits of the present invention include in various embodimentsone or more binding agents having a binding region and one or more tags,and an anti-tag antibody having an anti-tag region (e.g., an anti-tagantibody), as described herein.

The methods, compositions and kits described herein in certainembodiments include isolated polypeptide molecules that have beenengineered or isolated to act as binding agents or binding proteins. Abinding protein composition includes for example an amino acid sequenceselected from SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 20, 22, 24, 26, 28,30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52 or combinations thereof.In various embodiments, a binding protein composition includes anucleotide sequence that encodes an amino acid sequence, for example thenucleotide sequence is selected from SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13,19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51 orcombinations thereof. The bindings protein composition includes forexample a tag, for example a tag having an amino acid sequence of SEQ IDNO:15.

As used herein, the term “polypeptide” encompasses amino acid chains ofany length, including full length proteins (i.e., disease agents), inwhich the amino acid residues are linked by covalent peptide bonds. Apolypeptide comprises a portion of the binding agent, the entire bindingagent, or contains additional sequences. The polypeptides of the bindingagents of the present invention referred to herein as “isolated” arepolypeptides that are separated away and purified from other proteinsand cellular material of their source of origin. The compositions andmethods of the present invention also encompass variants of the abovepolypeptides and DNA molecules. A polypeptide “variant,” as used herein,is a polypeptide that differs from the recited polypeptide by having oneor more conservative substitutions and/or modifications, such that thefunctional ability of the binding agent to bind to the disease agenttarget is retained.

The present invention also encompasses proteins and polypeptides,variants thereof, or those having amino acid sequences analogous to theamino acid sequences of binding agents described herein. Suchpolypeptides are defined herein as analogs (e.g., homologues), ormutants or derivatives. “Analogous” or “homologous” amino acid sequencesrefer to amino acid sequences with sufficient identity of any one of theamino acid sequences of the present invention so as to possess thebiological activity (e.g., the ability to bind to the disease agenttarget). For example, an analog polypeptide can be produced with“silent” changes in the amino acid sequence wherein one, or more, aminoacid residues differ from the amino acid residues of any one of thesequence, yet still possesses the function or biological activity of thepolypeptide. The binding protein includes for example an amino acidhaving at least about 60% (e.g., 65%, 70%, 75%, 80%, 85%, 90% or 95%)identity or similarity with SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 20, 22,24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 56-87, 95 orcombination thereof. Percent “identity” refers to the amount ofidentical nucleotides or amino acids between two nucleotides or aminoacid sequences, respectfully. As used herein, “percent similarity”refers to the amount of similar amino acids between two amino acidsequences, i.e., having conservative amino acid changes compared to theoriginal sequences, or to the amount of similar nucleotides between twonucleotide sequences.

Referring to FIGS. 4 and 5, by comparing the B5 (SEQ ID NO: 24)polypeptide sequence to the other polypeptide sequences in the chart,the polypeptide sequence similarity is determined as follows: E-9 (SEQID NO: 38) is 74% similar, C5 (SEQ ID NO: 42) is 67% similar, B2 (SEQ IDNO: 40) is 68% similar, and F9 (SEQ ID NO: 44) is 73% similar. The BLASTwas done using default parameters on the NCBI website. Since these VHHshave been shown to compete with B5, i.e., for binding to the target, thepresent invention includes those sequences having a sequence similarityof at least about 65%. In like manner, by comparing the B5 (SEQ ID NO:23) nucleic acid sequence to the other nucleic acid sequences in thechart, the polypeptide sequence similarity is determined as follows: E-9(SEQ ID NO: 37) is 81% identical, C5 (SEQ ID NO: 41) is 75% identical,B2 (SEQ ID NO: 39) is 86% identical, and F9 (SEQ ID NO: 43) is 80%identical. The present invention includes those nucleic acid sequenceshaving a sequence identity of at least about 75%.

Homologous polypeptides are determined using methods known to those ofskill in the art. Initial homology searches are performed at NCBI bycomparison to sequences found in the GenBank, EMBL and SwissProtdatabases using, for example, the BLAST network service. Altschuler, S.F., et al., J. Mol. Biol., 215:403 (1990), Altschuler, S. F., NucleicAcids Res., 25:3389-3402 (1998). Computer analysis of nucleotidesequences can be performed using the MOTIFS and the FindPatternssubroutines of the Genetics Computing Group (GCG, version 8.0) software.Protein and/or nucleotide comparisons were performed according toHiggins and Sharp (Higgins, D. G. and Sharp, P. M., Gene, 199873:237-244, e.g., using default parameters). In certain embodiments, therecombinant multimeric binding protein acid sequence is an amino acidsequence that is substantially identical to sequences described herein,for example any of SEQ ID NOs: 56-87 and 95. The term “substantiallyidentical” is used herein to refer to a first amino acid sequence thatcontains a sufficient or minimum number of amino acid residues that areidentical to aligned amino acid residues in a second amino acid sequencesuch that the first and second amino acid sequences can have a commonstructural domain and/or common functional activity. For example, aminoacid sequences that contain a common structural domain having at leastabout 60% identity, or at least 75%, 85%, 95%, 96%, 98%, or 99%identity.

Calculations of sequence identity between sequences are performed asfollows. To determine the percent identity of two amino acid sequences,the sequences are aligned for optimal comparison purposes (e.g., gapscan be introduced in one or both of a first and a second amino acidsequence for optimal alignment). The amino acid residues atcorresponding amino acid positions or nucleotide positions are thencompared. When a position in the first sequence is occupied by the sameamino acid residue or nucleotide as the corresponding position in thesecond sequence, then the proteins are identical at that position. Thepercent identity between the two sequences is a function of the numberof identical positions shared by the sequences, taking into account thenumber of gaps, and the length of each gap, which need to be introducedfor optimal alignment of the two sequences.

The comparison of sequences and determination of percent identitybetween two sequences are accomplished using a mathematical algorithm.Percent identity between two amino acid sequences is determined using analignment software program using the default parameters. Suitableprograms include, for example, CLUSTAL W by Thompson et al., Nuc. AcidsResearch 22:4673, 1994 (www.ebi.ac.uk/clustalw), BL2SEQ by Tatusova andMadden, FEMS Microbial. Lett. 174:247, 1999(www.ncbi.nlm.nih.gov/blast/b12seq/b12.html), SAGA by Notredame andHiggins, Nuc. Acids Research 24:1515, 1996(igs-server.cnrs-mrs.fr/˜cnotred), and DIALIGN by Morgenstern et al.,Bioinformatics 14:290, 1998 (bibiserv.techfak.uni-bielefeld.de/dialign).

The methods, compositions and kits described herein in variousembodiments include nucleotide sequence or an isolated nucleic acidmolecule (encoding the binding protein) having a nucleotide sequence ofSEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 19, 21, 23, 25, 27, 29, 31, 33, 35,37, 39, 41, 43, 45, 47, 49, 51 or combinations thereof. See FIGS. 1, 3and 4. As used herein, the terms “DNA molecule” or “nucleic acidmolecule” include both sense and anti-sense strands, cDNA, genomic DNA,recombinant DNA, RNA, and wholly or partially synthesized nucleic acidmolecules. A nucleotide “variant” is a sequence that differs from therecited nucleotide sequence in having one or more nucleotide deletions,substitutions or additions. Such modifications are readily introducedusing standard mutagenesis techniques, such as oligonucleotide-directedsite-specific mutagenesis as taught, for example, by Adelman et al. (DNA2:183, 1983). Nucleotide variants are naturally occurring allelicvariants, or non-naturally occurring variants. Variant nucleotidesequences in various embodiments exhibit at least about 70%, morepreferably at least about 80% and most preferably at least about 90%homology to the recited sequence. Such variant nucleotide sequenceshybridize to the recited nucleotide sequence under stringent conditions.In one embodiment, “stringent conditions” refers to prewashing in asolution of 6×SSC, 0.2% SDS; hybridizing at 65° Celsius, 6×SSC, 0.2% SDSovernight; followed by two washes of 30 minutes each in 1×SSC, 0.1% SDSat 65° C. and two washes of 30 minutes each in 0.2×SSC, 0.1% SDS at 65°C.

The present invention also encompasses isolated nucleic acid sequencesthat encode the binding agents and in particular, those which encode apolypeptide molecule having an amino acid sequence of SEQ ID NOs: 2, 4,6, 8, 10, 12, 14, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44,46, 48, 50, 52, 56-87, 95 or combinations thereof.

As used herein, an “isolated” nucleotide sequence is a sequence that isnot flanked by nucleotide sequences which in nature flank the gene ornucleotide sequence (e.g., as in genomic sequences) and/or has beencompletely or partially purified from other transcribed sequences (e.g.,as in a cDNA or RNA library). Thus, an isolated gene or nucleotidesequence can include a gene or nucleotide sequence which is synthesizedchemically or by recombinant means. Nucleic acid constructs contained ina vector are included in the definition of “isolated” as used herein.Also, isolated nucleotide sequences include recombinant nucleic acidmolecules and heterologous host cells, as well as partially orsubstantially or purified nucleic acid molecules in solution. Thenucleic acid sequences of the binding agents of the present inventioninclude homologous nucleic acid sequences. “Analogous” or “homologous”nucleic acid sequences refer to nucleic acid sequences with sufficientidentity of any one of the nucleic acid sequences described herein, suchthat once encoded into polypeptides, they possess the biologicalactivity of any one of the binding agents described herein. Inparticular, the present invention is directed to nucleic acid moleculeshaving at least about 70% (e.g., 75%, 80%, 85%, 90% or 95%) identitywith SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 19, 21, 23, 25, 27, 29, 31, 33,35, 37, 39, 41, 43, 45, 47, 49, 51 or combinations thereof.

Also encompassed by the present invention are nucleic acid sequences,DNA or RNA, which are substantially complementary to the DNA sequencesencoding the polypeptides of the present invention, and whichspecifically hybridize with their DNA sequences under conditions ofstringency known to those of skill in the art. As defined herein,substantially complementary means that the nucleotide sequence of thenucleic acid need not reflect the exact sequence of the encodingoriginal sequences, but must be sufficiently similar in sequence topermit hybridization with nucleic acid sequence under high stringencyconditions. For example, non-complementary bases can be interspersed ina nucleotide sequence, or the sequences can be longer or shorter thanthe nucleic acid sequence, provided that the sequence has a sufficientnumber of bases complementary to the sequence to allow hybridizationtherewith. Conditions for stringency are described in e.g., Ausubel, F.M., et al., Current Protocols in Molecular Biology, (Current Protocol,1994), and Brown, et al., Nature, 366:575 (1993); and further defined inconjunction with certain assays.

The invention also provides vectors, plasmids or viruses containing oneor more of the nucleic acid molecules having the sequence of SEQ IDNO:1, 3, 5, 7, 9, 11, 13, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39,41, 43, 45, 47, 49, 51 or combinations thereof). Suitable vectors foruse in eukaryotic and prokaryotic cells are known in the art and arecommercially available or readily prepared by a skilled artisan.Additional vectors can also be found, for example, in Ausubel, F. M., etal., Current Protocols in Molecular Biology, (Current Protocol, 1994)and Sambrook et al., “Molecular Cloning: A Laboratory Manual,” 2nd ED.(1989).

Any of a variety of expression vectors known to those of ordinary skillin the art can be employed to express recombinant polypeptides of thisinvention. Expression can be achieved in any appropriate host cell thathas been transformed or transfected with an expression vector containinga DNA molecule that encodes a recombinant polypeptide. Suitable hostcells include prokaryotes, yeast and higher eukaryotic cells.Preferably, the host cells employed are E. coli, yeast, insect cells, ora mammalian cell line such as COS or CHO. The DNA sequences expressed inthis manner can encode any of the polypeptides described hereinincluding variants thereof.

Uses of plasmids, vectors or viruses containing the nucleic acids of thepresent invention include generation of mRNA or protein in vitro or invivo. In related embodiments, the methods, compositions and kitsencompass host cells transformed with the plasmids, vectors or virusesdescribed above. Nucleic acid molecules can be inserted into a constructwhich can, optionally, replicate and/or integrate into a recombinanthost cell, by known methods. The host cell can be a eukaryote orprokaryote and includes, for example, yeast (such as Pichia pastoris orSaccharomyces cerevisiae), bacteria (such as E. coli, or Bacillussubtilis), animal cells or tissue, insect Sf9 cells (such asbaculoviruses infected SF9 cells) or mammalian cells (somatic orembryonic cells, Human Embryonic Kidney (HEK) cells, Chinese hamsterovary cells, HeLa cells, human 293 cells and monkey COS-7 cells). Hostcells suitable in the present invention also include a mammalian cell, abacterial cell, a yeast cell, an insect cell, and a plant cell.

The nucleic acid molecule can be incorporated or inserted into the hostcell by known methods. Examples of suitable methods of transfecting ortransforming cells include calcium phosphate precipitation,electroporation, microinjection, infection, lipofection and directuptake. “Transformation” or “transfection” as used herein refers to theacquisition of new or altered genetic features by incorporation ofadditional nucleic acids, e.g., DNA. “Expression” of the geneticinformation of a host cell is a term of art which refers to the directedtranscription of DNA to generate RNA which is translated into apolypeptide. Methods for preparing such recombinant host cells andincorporating nucleic acids are described in more detail in Sambrook etal., “Molecular Cloning: A Laboratory Manual,” Second Edition (1989) andAusubel, et al. “Current Protocols in Molecular Biology,” (1992), forexample.

The host cell is maintained under suitable conditions for expression andrecovery of the polypeptides of the present invention. In certainembodiments, the cells are maintained in a suitable buffer and/or growthmedium or nutrient source for growth of the cells and expression of thegene product(s). The growth media are not critical to the invention, aregenerally known in the art and include sources of carbon, nitrogen andsulfur. Examples include Luria-Bertani broth, Superbroth, Dulbecco'sModified Eagles Media (DMEM), RPMI-1640, M199 and Grace's insect media.The growth media can contain a buffer, the selection of which is notcritical to the invention. The pH of the buffered Media can be selectedand is generally one tolerated by or optimal for growth for the hostcell.

The host cell is maintained under a suitable temperature and atmosphere.Alternatively, the host cell is aerobic and the host cell is maintainedunder atmospheric conditions or other suitable conditions for growth.The temperature is selected so that the host cell tolerates the processand is for example, between about 13-40° Celsius.

The invention having now been fully described, it is further illustratedby the following claims and by the examples, which are found in a paperpublished in the Public Library of Science (PLoS) One and entitled, “ANovel Strategy for Development of Recombinant Antitoxin TherapeuticsTested in a Mouse Botulism Model”, co-authored by Jean Mukherjee,Jacqueline M. Tremblay, Clinton E. Leysath, Kwasi Ofori, Karen Baldwin,Xiaochuan Feng, Daniela Bedenice, Robert P. Webb, Patrick M. Wright,Leonard A. Smith, Saul Tzipori, and Charles B. Shoemaker (12 pages;Mukherjee J et al. 2012 PLoS ONE 7(1): e29941.doi:10.1371/journal.pone.0029941). This published paper is herebyincorporated by reference herein in its entirety.

The following examples and claims are illustrative and are not meant tobe further limiting. Those skilled in the art will recognize or be ableto ascertain using no more than routine experimentation, numerousequivalents to the specific procedures described herein. Suchequivalents are within the scope of the present invention and claims.The contents of all references including issued patents and publishedpatent applications cited in this application are hereby incorporated byreference.

EXAMPLES Example 1 Toxins and Reagents

Botulinum neurotoxin serotype A1 (BoNT/A) and serotype B (BoNT/B) wereobtained from Metabiologics Inc. Each batch of toxin was calibrated toestablish the LD₅₀ dose in mice and stored in aliquots at −80° C. untiluse. Purified recombinant BoNT serotype A1 and B holotoxins containingmutations rendering them catalytically inactive (ciBoNTA, ciBoNTB)obtained. Sheep anti-BoNT/A1 antiserum was produced by immunization ofsheep with BoNT/A1 toxoid followed by BoNT/A1 holotoxin. Less than 1 μlof this sheep antitoxin serum protects mice from lethality whenco-administered with 10,000-fold the LD₅₀ of BoNT/A1. Reagents forWestern blotting were purchased from KPL (Gaithersburg, Md.).

C. difficile holotoxins TcdA and TcdB were generated by transformationof shuttle vectors pHis1522 (pHis-TcdA and pHis-TcdB respectively) intoB. megaterium described in Yang et al. 2008 BMC Microbiolgoy 8:192.Point mutations were introduced into conserved amino acids that areresponsible for binding to the substrate, uridine diphosphoglucose(UDP-Glucose), in order to generate GT-deficient holotoxins. To generateGT-mutant holotoxin A, a unique restriction enzyme (BamHI) site wasdesigned and constructed between sequences encoding GT and CPD domainsusing overlapping PCR. The primer sets used were:

pHis-F (5′- TTTGTTTATCCACCGAACTAAG -3′; SEQ ID NO: 90), Bam-R(5′- TCTTCAGAAAGGGATCCACCAG-3′; SEQ ID NO: 91), Bam-F(5′- TGGTGGATCCCTTTCTGAAGAC -3′; SEQ ID NO: 92), and Bpu-R(5′- ACTGCTCCAGTTTCCCAC -3′; SEQ ID NO: 93).

The final PCR product was digested with BsrGI and Bpu10I, and was usedto replace the corresponding sequence in pHis-TcdA. The resultingplasmid was designated pH-TxA-b. Sequences encoding triple mutations(W101A, D287N, and W519A) in the GT were synthesized by Geneart(Regensburg, Germany) and cloned into pH-TxA-b through BsrGI/BamHIdigestion. To generate the mutant holotoxin B construct, the sequencebetween BsrGI and NheI containing two point mutations (W102A and D288N)was synthesized and inserted into pHis-TcdB at the same restrictionenzyme sites, leading to a new plasmid pH-aTcdB. The mutant aTcdA andaTcdB were expressed and purified identical to the wild types in B.megaterium as described by Yang et al. 2008 BMC Microbiology 8:192. Thepurified aTcdA and aTcdB were used to immunize alpacas.

Example 2 Alpaca Immunization and VHH-Display Library Preparation

Purified, catalytically inactive mutant forms of full-length recombinantBoNT/A (ciBoNTA) and BoNT/B (ciBoNTB) proteins were obtained asdescribed in Webb et al. 2009 Vaccine 27: 4490-4497. Alpacas (twoanimals per immunization type) were immunized with either ciBoNTA orwith ciBoNTB. Additional alpacas were immunized with aTcdA or aTcdB. Theimmunization regimen employed 100 μg of protein in the primaryimmunization and 50 μg in three subsequent boosting immunizations atthree weekly intervals in aluminum hydroxide gel adjuvant in combinationwith oligodeoxynucleotides containing unmethylated CpG dinucleotides(alum/CpG; Superfos Biosector; Copenhagen, Denmark) adjuvant. Five daysfollowing the final boost immunization, blood from each animal wasobtained for lymphocyte preparation and VHH-display phage libraries wereprepared from the immunized alpacas as previously described (Maass etal. 2007 Int J Parasitol 37: 953-962 and Tremblay et al. 2010 Toxicon.56(6): 990-998). Independent clones (greater than 10⁶ total) wereprepared from B cells of alpacas successfully immunized with each of theBoNT immunogens.

Example 3 Anti-BoNT VHH Identification and Preparation

The VHH-display phage libraries were panned for binding to ciBoNTA orciBoNTB targets that were coated onto each well of a 12-well plate.Coating was performed by overnight incubation at 4° C. with one ml of a5 μg/ml target solution in PBS, followed by washing with PBS and twohours incubation at 37° C. with blocking agent (4% non-fat dried milkpowder in PBS). Panning, phage recovery and clone fingerprinting wereperformed as previously described (Ibid.). Based on phage ELISA signals,a total of 192 VHH clones were identified as strong candidate clones forbinding to BoNT/A, and 142 VHH clones were identified as strongpositives for binding to BoNT/B respectively. Of the strong positives,62 unique DNA fingerprints were identified among the VHHs selected forbinding to BoNT/A and 32 unique DNA fingerprints were identified forVHHs selected for binding to BoNT/B. DNA sequences of the VHH codingregions were obtained for each phage clone and compared for identifyinghomologies. Based on these data, twelve of the anti-BoNT/A VHHs andeleven anti-BoNT/B VHHs were identified as unlikely to have common Bcell clonal origins and were selected for protein expression. Expressionand purification of VHHs in E. coli as recombinant thioredoxin (Trx)fusion proteins containing hexahistidine was performed as previouslydescribed in Tremblay et al. 2010 Toxicon. 56(6): 990-998. Forheterodimers, DNA encoding two different VHHs were joined in framedownstream of Trx and separated by DNA encoding a fifteen amino acidflexible spacer having the amino acid sequence (GGGGS)₃. VHHs wereexpressed with a carboxyl terminal E-tag epitope. Furthermore, a numberof VHH expression constructions were engineered to contain a second copyof the E-tag by introducing the coding DNA in frame between the Trx andVHH domains. An example of a Trx fusion to a VHH heterodimer with twoE-tags is ciA-H7/ciA-B5(2E) shown in FIG. 13 panel C.

Example 4 VHH Target Binding Competition Analysis

Phage displaying individual VHHs were prepared and titered by phagedilution ELISA for recognition of ciBoNTA or ciBoNTB using HRP/anti-M13Ab for detection (Maass et al. 2007 Int J Parasitol 37: 953-962). Adilution was selected for each phage preparation that produced a signalnear the top of the linear range of the ELISA signal. The selected phagedilution (100 μl) for each VHH-displayed phage preparation were added to96 well plate that has been coated with ciBoNTA or ciBoNTB and thenpre-incubated for 30 minutes with 100 μl of a 10 μg/ml solutioncontaining a purified Trx/VHH fusion protein test agent or control inPBS. After an hour, the wells were washed and phage binding wasdetected. Test VHHs that reduced target binding of phage-displayed VHHsby less than two-fold compared to controls were considered to recognizedistinct epitopes. Positive controls were prepared in which the Trx/VHHcompetitor contained the same VHH as displayed on phage and typicallyreduced the ELISA signal detected by greater than 95%.

Example 5 Characterization of VHH Binding Properties

VHHs were tested for binding to native or atoxic mutant BoNT holotoxinsby standard ELISA using plates coated with 100 μl of 1 μg/ml protein.VHHs were also tested for recognition of BoNT subunits by ELISA usingplates coated with 5 μg/ml purified recombinant BoNT light chain or 1μg/ml BoNT heavy chain. See Tremblay et al. 2010 Toxicon. 56(6):990-998. VHHs were also characterized for recognition of subunits byWestern blotting on BoNT holotoxin following SDS-PAGE electrophoresisunder reducing conditions. VHHs were detected with HRP-anti-E-tag mAb(GE Healthcare) by standard procedures.

Example 6 Kinetic Analysis by Surface Plasmon Resonance

Assays to assess the kinetic parameters of the VHHs were performed usinga ProteOn XPR36 Protein. Interaction Array System (Bio-Rad, Hercules,Calif.) after immobilization of ciBoNT/A by amine coupling chemistryusing the manufacturer recommended protocol. Briefly, after activationof a GLH chip surface with a mixture of 0.4 Methyl (dimethylaminopropyl)carbodiimide (EDC) and 0.1 M N-hydroxysulfosuccinimide (sulfo-NHS)injected for 300 s at 30 μL/min, ciBoNT/A was immobilized by passing a60 μg/mL solution of the protein at pH 5 over the surface for 180 s at25 μL/min. The surface was deactivated with a 30 μL/min injection of 1 Methanolamine for 300 s. A concentration series for each VHH (between 2.5nM and 1000 nM, optimized for each antibody fragment) was passed overthe surface at 100 μL/min for 60 s, then dissociation was recorded for600 s or 1200 s. The surface was then regenerated with a 36 s injectionof 10 mM glycine, pH 2.0 at 50 μL/min. The running buffer used for theseassays was 10 mM Hepes, pH 7.4, 150 mM NaCl, 0.005% Tween-20. Data wasevaluated with ProteOn Manager software (version 2.1.2) using theLangmuir interaction model.

Example 7 BoNT Neutralization Assay Using Primary

Neuronal granule cells from the pooled cerebella of either 7-8 day oldSprague-Dawley rats or 5-7 day old CD-1 mice were harvested (Skaper etal 1979 Dev Neurosci 2: 233-237) and cultured in 24 well plates asdescribed by Eubanks et al 2010 ACS Med Chem Lett 1: 268-272. After atleast a week of culture the well volumes were adjusted to 0.5 mlcontaining various VHH dilutions or buffer controls followed immediatelyby addition of BoNT/A in 0.5 ml to a final 10 μM. After overnight at 37°C., cells were harvested and the extent of SNAP25 cleavage assessed byWestern blot as previously described (Eubanks, L. M. et al. 2007 Proc.Natl. Acad. Sci. USA 104: 2602-2607).

Example 8 Mouse Toxin Lethality Assay

Female CD1 mice (Charles River) about 15-17 g each were received fromthree to five days prior to use. On the day each assay was initiated,mice were weighed and placed into groups in an effort to minimizeinter-group weight variation. Appropriate dilutions of the VHH agentswere prepared in PBS. BoNT holotoxins were separately prepared in PBS atthe desired doses. Amounts (600 μl) of VHH agent and (600 μl) of thetoxin were combined and incubated at room temperature for 30-60 minutes.An amount (200 μl) of each mixture was administered by intravenousinjection at time point zero to groups of mice (five mice per group).Mice were monitored at least four times per day and assessed forsymptoms of toxin exposure and lethality/survival. Moribund mice wereeuthanized. Time to onset of symptoms and time to death were establishedfor each mouse.

Example 9 Mouse Toxin Lethality Assay with Agents AdministeredPost-Intoxication

Groups of mice were prepared as described in the description of themouse toxin lethality assay. Subjects were administered 10 LD₅₀ ofBoNT/A by intraperitoneal injection. At indicated timespost-intoxication, mice were administered 200 ul of material (e.g., VHHmonomer or VHH heterodimer) in PBS by intravenous injection. Mice weremonitored for symptoms of intoxication and death as described herein.

Example 10 Single-Chain Fvs (ScFv) that Recognize and Bind BoNT/A

To improve therapies that involve multiple monoclonal antibodies (mAbs)by using small recombinant peptide, protein or polynucleotide agentsthat have the same binding specificity as the mAbs, each of therecombinant binding agents is produced containing the same epitopic tag.A single mAb that recognizes the epitopic tag is co-administered topatients with the binding agents. The different agents bind to the sametargets as the multiple mAbs and the anti-tag mAb binds to these agentsthrough the epitopic tag. This permits delivery of the same therapeuticeffect that is achieved with multiple mAb therapy, but requires only asingle mAb. If desired, mAbs of different isotypes, or polyclonalanti-tag antibodies, could be used therapeutically to deliver differentimmune effector activities.

A number of small recombinant protein agents were generated. They werecalled single-chain Fvs (scFvs) and were observed to recognize botulinumneurotoxin serotype A (BoNT/A). These scFvs are recombinant proteinsthat represent the antigen combining region of an immunoglobulin.Several anti-BoNT/A scFvs were produced and were purified. Each scFvcontains the amino acid sequence (GAPVPYPDPLEPR; SEQ ID NO: 15) near thecarboxyl terminus which is an epitopic tag referred to herein as“E-tag.” An scFvs (scFv#2) was shown to neutralize BoNT/A in acell-based toxin assay (IC50 ˜7 nM). A second scFv (scFv#7) had littleor no neutralization activity in the assay, and was found to bind toBoNT/A with high affinity (Kd ˜1 nM).

The scFvs were tested for their ability to protect mice from thebotulinum toxin BoNT/A by intravenous administration of the agents andtoxin. The two scFvs were administered individually or together, andwere given to mouse subjects with and without anti-E-tag mAb byintravenous administration. Each subject was administered a dose of 10LD₅₀ of BoNT/A (i.e., an amount of BoNT/A ten-fold the LD₅₀), five miceper group. The results are shown in Table 1.

TABLE 1 scFv administration with and without anti- tag antibodyalleviates toxin morbidity Agents Administered (dose) SurvivalObservations none 0% Death in less than a day scFv#2 (20 μg) 0% Deathdelayed about a day scFv#7 (20 μg) 0% Death delayed less than a dayscFv#2 (20 μg) + anti-E-tag 100%  Symptoms severe mAb (25 μg) scFv#7 (20μg) + anti-E-tag 0% Death delayed several mAb (25 μg) days scFv#2 (10μg) + scFv#7 100%  No symptoms (10 μg) + anti-E-tag mAb (25 μg)

The results shown in Table 1 clearly show that a BoNT/A neutralizingscFv (scFv#2) alone did not significantly protect mice from the toxin.Subjects survived (100%) following co-administration scFV#2 and mAb thatrecognizes an epitopic tag (E-tag) on the scFv. More importantly,co-administering two scFvs, each with E-tag, and anti-tag mAbdramatically improved the protective effect.

Subjects were administered 10 LD₅₀ and lower doses of the scFvs and theanti-E-tag mAb, and were analyzed for percent survival. Further, twoadditional non-neutralizing anti-BoNT/A scFvs (scFv#3 and scFv#21) weretested in combination with the neutralizing scFv#2. Whether theanti-E-tag mAb would function upon administration at a different siteand time than the toxin was also tested.

The results in Table 2 confirm those data herein and further show thatthe mAb specific for the epitopic tag does not have to be pre-mixed withthe scFv containing the epitopic tag to be effective. In fact, doseswere administered at different sites and times. Combinations of twoscFvs (each with E-tags) and the single anti-E-tag mAb, provided greaterprotection than with one scFv alone. This synergistic protective effectoccurred using different scFvs and was observed at significantly lowerdoses of the scFvs or mAb than used in the data observed in Table 1.

TABLE 2 Anti-E-tag mAbs administered separately protected subjects fromtoxin Agents Administered (dose) Survival Observations none  0% Death inabout a day scFv#2 (10 μg)  0% Death delayed about 2 days scFv#2 (10μg) + anti-E-tag 100% Symptoms moderate mAb (10 μg) (mAb administeredintraperitoneally) scFv#2 (10 μg) + anti-E-tag 100% Symptoms mild mAb(10 μg) scFv#2 (10 μg) + anti-E-tag 100% Symptoms mild mAb (2 μg) scFv#2(2 μg) + anti-E-tag 100% Symptoms moderate mAb (2 μg) scFv#2 (5 μg) +scFv#7 100% No symptoms (3 μg) + anti-E-tag mAb (10 μg) scFv#2 (1 μg) +scFv#7 100% No symptoms (1 μg) + anti-E-tag mAb (10 μg) scFv#2 (5 μg) +scFv#3 100% No symptoms (4 μg) + anti-E-tag mAb (10 μg) scFv#2 (5 μg) +scFv#21 100% No symptoms (3 μg) + anti-E-tag mAb (10 μg)

Examples herein tested whether combinations of three and four scFvs withanti-tag mAb protect subjects from an amount of BoNT/A 100-fold,1000-fold, or 10,000-fold the LD₅₀, i.e., 100 LD₅₀ BoNT/A, 1000 LD₅₀BoNT/A or 10,000 LD₅₀ BoNT/A.

The data shown in Table 3 demonstrate the excellent potency of a taggedbinding agent as an antitoxin. Specifically, completely protection ofsubjects from even mild symptoms of intoxication by 1,000 LD₅₀ wasobserved using combinations of three or four scFvs with anti-E-tag mAb.Subjects were protected from lethality from a 10,000 LD₅₀ dose with acombination of four scFvs, although moderate symptoms were observed. Theability to protect mice receiving up to 10,000 LD₅₀ of BoNT/A isequivalent to the highest level of protection reported with pools ofdifferent anti-BoNT/A mAbs (Nowakowski et al, Proc Natl Acad Sci USA,99:11346-50).

TABLE 3 Combinations of scFv protect from 100, 1000, and 10,000 foldLD₅₀ BoNT/A doses in presence of 10 μg of anti-E-tag mAb Additionalagents BoNT/A administered (dose) Survival Observations   100 LD₅₀ None 0% Death in less than a day   100 LD₅₀ scFv#2 (2 μg) + scFv#3 100% Nosymptoms (2 μg) + scFv#21 (2 μg)  1,000 LD₅₀ None  0% Death in less thana day  1,000 LD₅₀ scFv#2 (2 μg) + scFv#3 100% No symptoms (2 μg) +scFv#21 (2 μg)  1,000 LD₅₀ scFv#2 (2 μg) + scFv#3 100% No symptoms (2μg) + scFv#7 (2 μg) + scFv#21 (2 μg) 10,000 LD₅₀ None  0% Death in a fewhours 10,000 LD₅₀ scFv#2 (2 μg) + scFv#3  0% Death delayed one day (2μg) + scFv#21 (2 μg) 10,000 LD₅₀ scFv#2 (2 μg) + scFv#3 100% Moderatesymptoms (2 μg) + scFv#7 (2 μg) + scFv#21 (2 μg)

The next example tested efficacy of a binding agent containing twocopies of the epitopic tag. The anti-BoNT/A binding agent, scFv#7, wasengineered to contain another copy of the E-tag peptide. The E-tagpeptide was present on the carboxyl terminus of each scFv. An alteredversion of scFv#7 (called scFv#7-2E) was engineered to be identical toscFv#7 and to have an additional copy of the E-tag peptide fused to theamino terminus.

TABLE 4 Protection from BoNT/A using scFvs having multiple tag sequencesin presence of 10 μg of anti-E-tag mAb BoNT/A Additional agents LD₅₀administered (1 μg each) Survival Observations 100 None 0% Death in lessthan 6 hours 100 scFv#2 + scFv#3 + 100%  No symptoms scFv#7 100 scFv#2 +scFv#3 + 100%  No symptoms scFv#7-2E 1,000 None 0% Death in less than 2hours 1,000 scFv#2 + scFv#3 + 0% Death delayed 2 days scFv#7 1,000scFv#2 + scFv#3 + 100%  No symptoms scFv#7-2E 10,000 None 0% Death inless than 2 hours 10,000 scFv#2 + scFv#3 + 0% Death delayed less thanscFv#7 a day 10,000 scFv#2 + scFv#3 + 20%  Death delayed many scFv#7-2Edays 10,000 scFv#2 + scFv#3 + 0% Death delayed 2 days scFv #21 + scFv#710,000 scFv#2 + scFv#3 + 100%  Moderate symptoms scFv #21 + scFv#7-2E

The results in Table 4 demonstrate that the binding agent with twoepitope tags dramatically improved the in vivo antitoxin efficacy of thetagged binding agent. With a combination of three scFvs, includingseFvs#2, scFvs#3 and scFvs#7 or scFvs#7-2E, clearly the use ofscFvs#7-2E was substantially superior in protection of mice to the useof scFvs#7 with only one E-tag. The improvement by presence of twocopies of tag was particularly evident in the groups of mice challengedwith 1,000 LD₅₀. In these groups, the triple combination ofscFv#2+scFv#3+scFv#7 was insufficient to allow survival of the mice.When scFv#7 was replaced with scFv#7-2E, all the mice survived withoutsymptoms. Furthermore, use of a pool of scFv#2+scFv#3+scFv#7-2Epermitted the survival of one of five mice challenged with 10,000 LD₅₀and delayed the death of the other mice by several days. The equivalentpool with scFv#7 having only one E-tag only delayed death for one day inmice challenged with 10,000 LD₅₀. Finally, an identical combination offour scFvs (#2, #3, #21 and #7) in which the efficacy using scFv#7 wascompared with scFv#7-2E. Administering only one μg of each scFv, thepresence of scFv#7 did not result in survival of mice challenged with10,000 LD₅₀, and the same combination the scFv#7-2E was protective.These data show that mice were effectively protected from high doses oftoxin by administering a smaller number high affinity binding agents,each containing two or more epitope tags together with an anti-tag mAb.

The method herein improves therapeutic agent flexibility, provideshighly stable binding agents with long shelf life, substantially reducesthe cost of production, and permits commercially feasible therapeuticapplications that involve multiple target agents. Furthermore, thestrategy herein will permit much more rapid development of newantitoxins. The binding agents are much more quickly developed tocommercialization than mAbs. The single anti-tag mAb needed forco-administration is the same for therapies requiring different taggedbinding agents and thus can be pre-selected for its commercial scale upproperties and stockpiled in advance of the development of the bindingagents.

An immediate application is in anti-toxin therapy, an area of highinterest because of bioterrorist threats. For example, it is now thoughtthat effective prevention of botulinum intoxication using toxinneutralizing mAbs will require administration of three different mAbseach targeting the same toxin. Since there are at least seven differentbotulinum toxins, this suggests that 21 different mAbs (or more) mayneed to be stockpiled for use in the event of a major botulism outbreakas might occur through bioterror. Monoclonal antibodies are veryexpensive to produce and have relatively short shelf lives. Methods andcompositions herein would make it possible to produce 21 differentrecombinant binding agents, each having longer shelf-life and lowerproduction costs, and then stockpile only a single mAb. It is possiblethat this approach could open up many other mAb therapeutic strategiesthat involve multiple binding targets, but which have not been pursuedbecause of prohibitive development and production costs and poor productshelf life. Methods and compositions herein permit the use of mAbs ofdifferent antibody isotypes to be used with the same binding agents toprovide greater therapeutic flexibility.

Example 11 BoNT/A VHHs Binding Agents

VHH binding agents were identified, produced and purified that werespecific to each of botulinum neurotoxin serotype A (BoNT/A) andserotype B (BoNT/B). The VHHs made herein included nine amino acids atthe amino coding end and which are associated with the forward PCRprimer sequence. See FIG. 3 panels A-C for the sequences. Thesesequences derive from ‘framework 1’ and include minor variants of theoriginal coding sequence. The most common amino acid sequence isQVQLVESGG (SEQ ID NO: 16) and which is the amino acid sequence used inassays shown in FIG. 3 panels A-C.

At the carboxyl coding end of the VHHs either amino sequence, AHHSEDPS(SEQ ID NO: 17), or the amino sequence, EPKTPKPQ (SEQ ID NO: 18) islocated, present in the VHHs sequence as shown in FIG. 3 panels A-C, andthese were observed to be interchangeable without loss of function.Identical clones were identified from alpacas that vary only in thehinge sequence and retain virtually the same target binding function.See also D. R. Maass et al. 2007 Journal of Immunological Methods324:13-25.

As a result of the altered splicing, the amino acid sequence that joinsthe VH domain to the CH2 domain in heavy chain IgGs is called the“hinge” region, and is unique to this class of camelid antibodies (SeeD. R. Maass et al. 2007 Journal of Immunological Methods 324:13-25 whichis incorporated by reference in its entirety). The two distinct hingesequence types found in camels and llamas are referred to as the “short”hinge and the “long” hinge respectively. SEQ ID NO: 17 is a short hingesequence derived from a camel, and SEQ ID NO: 18 is a long hingesequence derived from a llama.

During screening for VHH binding agents, different coding sequences areidentified that display significant homology among randomly identifiedclones. VHH sequences that are homologous are predicted to be relatedand thus to recognize the same epitope on the target to which they havebeen shown to bind. Examples herein experimentally demonstrate epitoperecognition by methods for binding competition. These findingsdemonstrate that significant variation is permitted in VHH amino acidsequences without loss of target binding. An example of the extent ofvariation permitted is shown in FIG. 4 panels A-B. Each VHH identifiedin FIG. 4 panels A-B as a BoNT/A binder was experimentally observed tobind to the same epitope as JDQ-B5 based on binding competition assays.

FIG. 5 shows a phylogenetic tree that compares the homology among BoNT/Abinding VHHs within the JDQ-B5 competition group to random alpaca VHHs.The homology comparison uses the unique amino acids that are presentbetween the forward PCR primer sequences and the hinge region (above).The distance of the lines is a measure of homology; the shorter thedistance separating two VHHs, the more homologous. Four VHHs that bindto the same epitope as JDQ-B5 cluster within a group that is distinctfrom the random VHHs as shown, strong evidence of relatatedness of theseclones. The results show that substantial variation in the VHH sequenceis tolerated without loss of the target binding capability.

The coding DNAs for two different VHH monomers were cloned in an E. coliexpression vector in several different ways to produce differentrecombinant proteins. To produce single VHH monomers, the VHH coding DNAwas inserted into the plasmid pET32b to fuse the VHH in frame with anamino terminal bacterial thioredoxin and a carboxyl terminal epitopictag (E-tag GAPVPYPDPLEPR; SEQ ID NO: 15). Additional coding DNA derivingfrom the pET32b expression vector DNA was also present between thethioredoxin and VHH coding sequences, the DNA encoding six histidines(to facilitate purification) and an enterokinase cleavage site, DDDDK topermit enzymatic separation of thioredoxin from the VHH. The resultingexpression vectors were used for expression of VHH monomers. VHHmonomers JDQ-H7 (SEQ ID NO: 32, referred to as “H7) and JDQ-B5 (SEQ IDNO: 24, referred to as “B5”) were expressed using this system (FIG. 6).A representation of the two monomer VHH proteins produced by theseexpression vectors, labeled H7/E and B5/E, is shown in FIG. 10 panel A.

Expression vectors were prepared in pET32b in which DNA encoding twoiterations of the VHH monomer (e.g., SEQ ID NOs: 46 and 48) was present,and the monomers joined in frame to yield heterodimers. For theseconstructions, the two nucleic acid sequences encoding the VHHs wereseparated by a nucleotide sequence encoding a 15 amino acid linker, SEQID NO: 55, that provides a flexible spacer (fs) between the expressedVHH proteins to separate the domains and facilitate independent folding.The E-tag coding DNA followed the second VHH coding DNA (SEQ ID NO: 49)in frame to obtain a single-tagged VHH heterodimer H7B5/E (SEQ ID NO:50), expression of which is shown in FIG. 10 panel B. A second copy ofthe E-tag coding DNA (e.g., SEQ ID NO: 51) was included upstream of thefirst VHH (at the amino coding end) for expression of a double-taggedVHH heterodimer E/H7/B5/E (SEQ ID NO: 52) shown in FIG. 10 panel B.

The thioredoxin fusion partner was included to improve expression andfolding of the VHHs, and was observed as not necessary for VHH function.The activity of the VHH agents to protect mice from BoNT/A intoxicationin mouse lethality assays was tested using VHH agents in whichthioredoxin was cleaved (by enterokinase) from the VHH. It was observedthat absence of thioredoxin caused no significant reduction in activity.

A single-tagged heterodimer VHH was predicted to lead to decoration ofthe BoNT toxin by the anti E-Tag mAb in a ratio of 1:1. Accordingly, asingle-tagged heterodimer was expected to bind at two sites on the toxinand lead to decoration of the toxin with two anti E-tag antibodies (seeFIG. 7). A double-tagged heterodimer provides for binding of the antiE-tag antibody in a ratio of 2:1 and thus should bind at two sites onthe toxin and lead to decoration of the toxin with four anti-tagantibodies (see FIG. 8). These agents were tested for their ability toprotect mice from BoNT/A toxin.

For these examples, the VHH agents and the toxin were pre-mixed and thenintravenously administered to groups of five subjects (mice) per group.The subjects were monitored and the time to death was noted for thosethat succumbed to the toxin. In the results shown in FIG. 9 panel A, apool of two VHH monomers, H7/E and B5/E (1 μg of each monomer persubject), in the presence of anti-E-tag mAb (Phadia, Sweden) (5μg/mouse) delayed death only about one day in mice exposed to 1,000 LD₅₀of BoNT/A. The single-tagged VHH heterodimer, H7/B5/E (2 μg/mouse) inthe presence of anti-E-tag mAb (5 μg/mouse) delayed death by about a dayand a half in mice exposed to 1,000 LD₅₀ of BoNT/A.

In contrast, it was observed that the double-tagged heterodimer,E/H7/B5/E (2 μg/mouse) administered with anti-E-tag mAb resulted in fullsurvival of mice exposed to 1,000 LD₅₀ and even 10,000 LD₅₀ of BoNT/A(FIG. 9 panel B). Mice given the double-tagged VHH heterodimer,E/H7/B5/E, in the absence of co-administered anti-E-tag mAb, did notsurvive a 1,000 LD₅₀ amount of BoNT/E, showing that the anti-tag mAb wasnecessary for full efficacy. The ability of the double-tagged VHHheterodimer, E/H7/B5/E, administered with anti-E-tag mAb to protect miceagainst 10,000 LD₅₀ demonstrates that this treatment achieved a level ofefficacy similar to that obtained with a commercial polyclonal antitoxinsera.

In other examples, the BoNT/A-binding VHH heterodimer agents were testedfor their ability to prevent death in subjects previously exposed toBoNT/A. In these examples, groups of five subjects were first exposed to10 LD₅₀ BoNT/A. Then after 1.5 or three hours from exposure mice weretreated either with the E/H7/B5/E heterodimer agent (2 μg/subject)administered with anti-E-tag mAb (5 μg/subject), or with a dose ofpotent polyclonal anti-BoNT/A sera that had been prepared in sheep. Thissera had been previously shown to protect subjects against 10,000 LD₅₀of BoNT/A when it was co-administered with the toxin (assays performedas in previous paragraph). All subjects were monitored and the time todeath was determined for non-survivors. Control subjects (2 groups offive each) died within about a day. All subjects treated with polyclonalantisera 1.5 hour post-intoxication (five) survived, and four of fivesubjects treated three hours post-exposure both 1.5 hours and threehours post-intoxication survived. Five out of five subjects treated withthe VHH heterodimer and anti-E-tag mAb both 1.5 hours and three hourspost-exposure survived. Thus the VHH heterodimer and anti-E-tagtreatment was at least as effective as conventional polyclonalantitoxins at protecting subjects from BoNT exposure in the moreclinically relevant post-exposure challenge model.

Example 12 Neutralization of Botulinum Neurotoxin Using VHH BindingProteins

Examples herein show that scFv antitoxin compositions preventdevelopment of disease symptoms in subjects exposed to a botulinumtoxin. These antitoxin agents were antibodies that bound the toxin andneutralized the activity of the toxin and/or promoted rapid clearancefrom the body. Data show that effective neutralization was achievedusing a mixture of two high-affinity toxin VHH binding agents, each ofwhich strongly neutralized toxin in cell-based assays. Administration ofa multimeric composition was much more effective at protecting subjectsfrom toxin than a pool of two neutralizing monomer binding agents only.

Camelid heavy chain only Vh domain (VHH) binding agents with highaffinity for Botulinum neurotoxin serotype A (BoNT/A) were producedincluding H7 (SEQ ID NO: 56), B5 (SEQ ID NO: 57). Methods of generatingVHH binding agents are shown in Shoemaker et al. U.S. application Ser.No. 12/032,744 which is application 2010/0278830 A1 published Nov. 4,2010, and Shoemaker et al. U.S. application Ser. No. 12/899,511 which isapplication 2011/0129474 A1 published Jun. 2, 2011, each of which isincorporated herein by reference in its entirety.

VHHs (H7, B5 and C2) displayed potent BoNT/A neutralization activity inassays of exposure or intoxication of primary neurons in culture. The H7VHH and B5 VHH monomers were expressed in E. coli and a singleheterodimeric polypeptide (H7/B5) was constructed and expressed with theH7 and B5 VHH domains/subunits separated by a fifteen amino acidflexible spacer having three repeats of amino acid sequence GGGGS (SEQID NO: 55). A combination of the H7 monomer binding agent and B5 monomerbinding, and a H7/B5 single chain heterodimer binding agent were testedto determine ability to protect mouse subjects from death caused byBoNT/A. The subjects received ten-fold the lethal dose of BoNT/A thatcauses death in 50% of mice (10 LD₅₀), and either 1.5 hours or threehours later were administered either: 1 micrograms (μg) of H7 bindingagent; a sheep antitoxin serum produced against BoNT/A; 1 μg of B5monomer binding agent; or 2 μg of H7/B5 single chain heterodimer bindingagent (FIG. 11 panels A-B). The amount of sheep antitoxin serumadministered was equivalent to the amount of commercial antitoxin serumgenerally administered.

Data show that subjects administered a combination of monomeric H7 andB5 binding agents died within three days. Control subjects administeredno therapeutic agent died within one day (FIG. 11 panels A-B). Subjectsadministered the sheep antitoxin serum survived at 80%. Most important,subjects administered H7/B5 single chain heterodimer binding agentsurvived additional days compared to the control subjects, with 80% ofsubjects administered H7/B5 heterodimer binding agent surviving forseven days.

Example 13 Neutralization of C. difficile Toxins Using HeteromultimerBinding Agents

A set of VHH binding agents that bind Clostridium difficile toxin B(TcdB) were obtained and shown in Examples herein to inhibit the abilityof the toxin to intoxicate or infect cells (FIG. 12 panel A). Potentanti-TcdB neutralizing VHHs were selected, identified by codes names 5Dand E3, and were expressed as separate monomers or as a heterodimer. Apool/mixture of VHH monomers, 5D and E3, was compared in for ability toprevent TcdB lethality to cells to the 5D/E3 heterodimer.

CT26 cells were exposed to TcdB (100 picograms/ml) in the presence ofdifferent concentrations (0.03 nM, 0.1 nM, 0.3 nM, 1 nM, 3 nM, 10 nM, or30 nM) of: a mixture of 5D VHH monomer (SEQ ID NO: 67) and E3 VHHmonomer (SEQ ID NO: 68), or a 5D/E3 heterodimer (SEQ ID NO: 87). Controlcells were not administered neutralizing agents. Cell rounding caused byTcdB was monitored using a phase-contrast microscope.

Culture media from expressing cells were administered with either themixture of 5D and E3 VHH monomers, or the 5D/E3 VHH heterodimer werefound to be effective in protecting the cells from TcdB associated cellrounding. Control cells (100%) showed cell rounding and negative indiciaof TcdB following toxin exposure.

It was observed that administering 0.1 nM 5D/E3 heterodimer to subjectsprior to TcdB exposure resulted in 50% cell rounding (i.e., 50% TcdBinfection; FIG. 12 panel B). The same level cell rounding protection(50%), was achieved with 1 nM of the mixture of 5D and E3 monomers.Thus, the 5D/E3 VHH heterodimer was observed to be about ten-fold morepotent as a toxin neutralizing agent than a pool containing the same twoVHHs as monomers (FIG. 12 panel B).

The improved antitoxin and protective potency 5D/E3 heterodimer wasfurther analyzed using an in vivo toxin challenge mouse model. Subjectswere co-administered a lethal dose of TcdB (1 ng/mL) with either amixture of 500 nanograms (ng) of 5D monomer and 500 ng E3 VHH monomer;or with 250 ng of 5D/E3 VHH heterodimer; or with phosphate bufferedsaline, PBS. See FIG. 12 panel C. See Data show that each of the VHHbinding agents was a more effective TcdB neutralizing agent for subjectsthan the PBS control. Survival was observed at 100% for subjectsadministered 5D/E3 VHH heterodimer (250 ng) and at about 40% forsubjects administered a mixture of 5D and E3 VHH monomers. Controlsubjects receiving PBS survived at a rate of 20%.

Data show that subjects administered a mixture of 5D and E3 monomerssurvived for fewer days and were less protected from a lethal TcdBchallenge than subjects administered the 5D/E3 heterodimer (FIG. 12panel C). Most important the improved protection and neutralizingability of the 5D/E3 heterodimers was observed even if the amount ofheterodimer administered was 75% less than the amount of the mixture of5D and E3 monomers. Further analysis was performed in Examples below todetermine the relative factors for VHH monomers and heterodimers toeffectively neutralize and clear disease agent targets from the body(FIG. 12 panels A-C).

Example 14 Identification and Characterization of Anti-BoNT VHHs

Serum clearance of Botulinum neurotoxin serotype A (BoNT/A) wasdramatically accelerated by administering a pool of differentepitopically-tagged single-chain Ig variable fragment (scFv) domainbinding agents with an anti-tag monoclonal antibody (Shoemaker et al.U.S. Ser. No. 12/032,744 application 2010/0278830 A1 published Nov. 4,2010; Shoemaker et al. U.S. Ser. No. 12/899,511 application 2011/0129474A1 published Jun. 2, 2011; Sepulveda et al. 2009 Infect Immun 78:756-763, and Tremblay et al. 2010 Toxicon. 56(6): 990-998, each of whichis incorporated herein in its entirety).

To determine whether a more commercially and clinically acceptablebinding agent than scFvs could be identified, a panel of camelidheavy-chain-only Vh (VHH) binding agents having high affinity forepitopes of BoNT/A holotoxin was produced. VHHs were obtained that boundto an epitope of a distinct BoNT serotype, BoNT/B holotoxin, and theseVHHs were tested for antitoxin efficacy. Competition ELISAs wereperformed to identify the VHHs with the highest affinity for uniqueepitopes on BoNT/A and BoNT/B. VHHs specific for each of BoNT/A (FIG. 13panel A) and for BoNT/B (FIG. 13 panel B) were identified.

The VHHs in FIG. 13 panels A-B include amino acid sequence QLQLVE (SEQID NO: 88) and QVQLVE (SEQ ID NO: 89) at the amino terminus region. Thesequence was encoded by the PCR primer used to generate the VHH-displaylibrary (Maass et al. 2007 Int J Parasitol 37: 953-962). The eight aminoacids shown at the carboxy-terminus end were encoded by the short hingeor long hinge PCR primers that were used to generate the VHH library.

The amino acid sequences for double-tagged VHH heterodimer antitoxinsthat specifically bind BoNT/A: ciA-H7/ciA-B5(2E) and ciA-F12/ciA-D12(2E)are shown in FIG. 13 panel C. Each heterodimer included two VHH monomersand two epitopic tags. The amino acid sequences of the tags within theamino acid sequences of the heterodimers are underlined (FIG. 13 panelC). The amino acid sequence preceding the first E-tag in each VHHprotein contained the thioredoxin fusion partner and hexahistidineencoded by the pET32b expression vector. The VHH sequences were flankedby the two E-tag peptides and were separated by the unstructured spacerhaving amino acid sequence (GGGGS)₃, SEQ ID NO: 55.

Each VHH was purified from E. coli as a thioredoxin fusion proteincontaining a single carboxyl-terminal epitopic tag (E-tag). Sodiumdodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysesof VHH monomers and VHH heterodimers was performed (FIG. 14 panels A-B).The channels were loaded with one microgram (μg) of each VHH monomer orheterodimer. Molecular weight markers (12, 31, 45, 66, 97, 116 and 200kilodaltons) are shown on the border of each gel. FIG. 14 panel A showsSDS-PAGE analysis of the tagged (E) VHH monomers: ciA-D1, ciA-H4,ciA-H11, ciA-A5, ciA-C2, ciA-D12, ciA-F12, ciA-G5, and ciA-H7. Darkbands were observed at approximately 35-38 kilodalton molecular weightfor all single tagged VHH monomers Channels loaded with ciA-D1, ciA-H4,ciA-H11, and ciA-B5 showed light bands at about 45-46 kilodaltons (kDa),and at about 59 kDa to about 62 kDa molecular weight. SDS-PAGE analysiswas performed also on single- or double-tagged VHH heterodimers:ciA-H7/ciA-B5 singly tagged on ciA-B5; double tagged ciA-H7/ciA-B5having a tag on both ciA/H7 and ciA-B5, ciA-F12/ciA-D12 singly tagged onciA-B5; double tagged ciA-F12/ciA-D12 having a tag on both ciA/F12 andciA-D12, double tagged ciA-A11/ciA-B5 having a tag on both ciA/A11 andciA-B5 (FIG. 14 panel B). Strong dark bands at about 48 kDa to about 58kDa molecular weight were observed for each heterodimer (FIG. 14 panelB).

The unique BoNT/A binding VHHs were further characterized and analyzedfor ability to affinity target BoNT/A using surface plasmon resonance(SPR) in which a lower Kd indicates stronger binding/affinity betweenthe VHH and the toxin target. Analysis was performed also to determinethe ability of the BoNT/A binding VHHs to prevent intoxication ofprimary neurons in culture (FIG. 15 and Table 5).

Neuronal granule cells from pooled cerebella of seven day old to eightday old Sprague-Dawley rats or five day old to seven day old CD-1 micewere harvested as described by Skaper et al 1979 Dev Neurosci 2:233-237.The cells were then cultured in 24-well plates as described by Eubankset al 2010 ACS Med Chem Lett 1: 268-272. After a week or more ofculture, each culture well was adjusted to a volume of 0.5 ml withdilutions of VHHs (ciA-H7, ciA-B5, ciA-C2, ciA-D12, ciA-F12, ciA-A5 orciA-G5) or a buffer control, and BoNT/A (ten picomoles) was added. Afterovernight incubation at 37° C., cells were harvested and the extent ofsynaptosomal-associated protein 25 (SNAP25) cleavage was determined byWestern blot using commercially available rabbit anti-SNAP25 (Sigma).See FIG. 15. SNAP-25 is a membrane bound protein anchored to thecytosolic face of membranes by palmitoyl side chains within the moleculethat is involved in the regulation of neurotransmitter release.Botulinum toxin serotypes including serotypes A, C and E function tocleave SNAP-25, resulting in paralysis and clinically developedbotulism.

The upper band shown in the Western blot photographs is uncleavedSNAP25, and the lower band indicates cleaved SNAP25 (FIG. 15). SNAP25cleavage (i.e., presence of a lower band) resulting from exposure tobotulinum toxin was observed. VHHs were identified by the criterion thatat concentrations of less than 0.1 nanomoles (nM) were observed toinhibit BoNT/A cleavage of SNAP25 (i.e., no lower band), are strongneutralizing agents. Weak neutralizing VHHs were identified as VHHs thatrequired greater than 1 nM to inhibit BoNT/A cleavage of SNAP25. VHHsthat required greater than 10 nM to prevent SNAP25 cleavage wereidentified as having no toxin neutralizing ability (FIG. 15).

It was observed that about equimolar amounts of ciA-B5, ciA-C2 andciA-H7 VHHs prevented intoxication of neurons with 10 picomoles ofBoNT/A. Two VHHs (ciA-D12 and ciA-F12) were observed to have no ornegligible toxin neutralizing activity even at 1,000-fold excess VHH totoxin. Two VHHs (ciA-A5 and G5) displayed intermediate neutralizingactivity compared to ciA-B5, ciA-C2 and ciA-H7, the stronglyneutralizing VHHs, and ciA-D12 and ciA-F12, the non-neutralizing VHHs(FIG. 15 and Table 5).

Thus, ciA-B5, ciA-C2 and ciA-H7 were determined to be strongneutralizing VHHs. Other isolates including ciA-D12 and ciA-F12 wereobserved to be non-neutralizing VHHs that produced no detectable toxinneutralization.

Example 15 Protection from BoNT/A Lethality Using Monomeric Anti-BoNT/AVHHs

Epitopically tagged anti-BoNT/A VHH compositions were shown in theExample herein to prevent toxin induced lethality in the presence orabsence of the clearing anti-tag mAb. Methods of testing VHHs are shownin Sepulveda et al. 2009 Infect Immun 78:756-763, and Tremblay et al.2010 Toxicon. 56(6): 990-998. Pools/mixtures of two, three, four or sixdifferent anti-BoNT/A VHH monomers with or without anti-E-tag clearingantibody were co-administered to subjects with an amount (1000 LD₅₀ or10,000 LD₅₀) of BoNT/A holotoxin. Subjects were then monitored forsymptoms of toxin lethality and were observed for time to death.

The subjects were co-administered BoNT/A with either a mixture of ciA-H7and ciA-B5 monomers, or a mixture of ciA-D12 and ciA-F12 monomers (FIG.16 panel A bottom graphs). Each mixture was administered with (+αE) orwithout (−αE) anti-E-tag clearing antibody that specifically bound theepitopic tags located on the VHHs. Control subjects were administeredtoxin only. Unless indicated otherwise, a dashed line in FIGS. 16-24indicates that no anti-E-tag antibody was administered to the subjects.Each monomeric VHH was used at a total dose of two micrograms (μg) permouse to ensure that the only the complexity and/or identity of the VHHmixtures was varied among groups and was the cause of observed antitoxinefficacy.

Results obtained show that subjects administered ciA-D12 and ciA-F12,two anti-BoNT/A VHH monomers previously determined not to neutralizeBoNT/A in cell assays, did not survive toxin challenge for any greatertime than did control subjects administered toxin only (FIG. 16 panel Abottom graphs). Administration of 5 μg amounts of anti-E-tag clearingantibody (αE) to subjects only slightly prolonged time before death.Data show that subjects administered neutralizing VHH monomers ciA-H7and ciA-B5 with anti-E-tag clearing antibody were slightly protectedagainst BoNT/A compared to subjects administered ciA-D12 and ciA-F12,and anti-E-tag clearing antibodies. Thus, the decoration of BoNT/A withtwo clearing antibodies provided little or no therapeutic benefit to thesubjects.

Administration to subjects of a mixture of ciA-B5, and ciA-H7 monomersabsent clearing antibody only delayed time to death. Data show thatsubjects challenged with 100-fold the LD₅₀ of BoNT/A (approximately 5nanograms total) survived longer following administration of a mixtureof neutralizing ciA-B5 and ciA-H7 compared to control subjectsadministered no VHHs. Most important, it was observed thatco-administration of clearing antibody and the neutralizing VHHsresulted in 100% survival of subjects challenged with 100-fold the LD₅₀of BoNT/A (FIG. 16 panel A bottom left graph). At a challenge of1,000-fold the LD₅₀ of BoNT/A, death was delayed about one additionalday for subjects co-administered a mixture of ciA-B5 and ciA-H7 andanti-E-tag clearing antibody compared to subjects administered VHHsalone or control subjects (FIG. 16 panel A bottom right graph). Thus, itwas observed that administering a mixture of toxin neutralization VHHmonomers with clearing antibody provided greater therapeutic benefit andprotection against BoNT/A than administering VHHs absent the clearingantibody. Relative affinity of each VHH influences the therapeuticeffect of the VHH, likewise for VHHs having similar sub-nanomolaraffinities (See Table 5).

Whether mixtures of VHH monomers containing both neutralizing VHHs andnon-neutralizing VHHs were effective antitoxin agents was furthertested. Subjects were co-administered 1,000-fold or 10,000-fold BoNT/ALD₅₀ and one VHH monomer mixture of either a mixture of ciA-B5, ciA-H7,and ciA-C2; or a mixture of ciA-H7, ciA-A5 and ciA-D12 with (+αE) orwithout (−αE) an anti-E-tag clearing antibody preparation thatspecifically binds the epitopic tags located on the VHHs (FIG. 16 panelB bottom graphs). Control subjects were administered toxin only.

Administration of a mixture of ciA-B5, ciA-H7, ciA-C2 monomers, eachcapable of potent toxin neutralization, delayed death less than a day inmice exposed to 1000-fold the LD₅₀ of BoNT/A (FIG. 16 panel B bottomleft graph). Subjects were completely protected (100% survival) at1000-fold the LD₅₀ of BoNT/A following administration mixture of ciA-B5,ciA-H7, and ciA-C2 monomers and clearing antibody. Co-administration of10,000-fold the LD₅₀ of BoNT/A (a total amount of 0.5 μg), a mixture ofciA-B5, ciA-H7, ciA-C2 monomers and clearing antibody delayed death morethan two days in subjects (See FIG. 16 panel B bottom right graph)compared to control subjects.

TABLE 5 SPR binding data for VHH monomers and heterodimers clone proteinepitope^(#) neutralization* SPR Kd (nM) subunit{circumflex over ( )}Genbank JDY-33 ciA-H7 A1 strong 0.06 +/− 0.07 Lc HQ700708 JDT-2 ciA-D1A1 strong  0.71 +/− 0.004 Lc JEC-3 ciA-H4 A1 not done 1.54 +/− 0.06 LcJEC-11 ciA-H11 A1 not done  4.3 +/− 0.09 Lc JDY-46 ciA-C2 A2 strong 2.7+/− 3.1 Lc HQ700705 JDY-9 ciA-B5 A3 strong 0.17 +/− 0.06 Hc HQ700704JED-27 CiA-F12 A4 none 0.24 +/− 0.03 Lc HQ700706 JDU-26 ciA-D12 A5 none0.21 +/− 0.1  Lc HQ700702 JDY-2 ciA-A5 A6 weak 1.05 +/− 0.05 noneHQ700703 JDY-59 ciA-G5 A7 weak 0.32 +/− 0.03 none HQ700707 JFA-10CiB-H11 B1 not done 0.26 +/− 0.01 none JFX-30 CiB-A11 B2 not done 0.84+/− 0.68 none JFV-48 ciB-B5 B3 not done 0.97 +/− 0.04 none JFV-40 ciB-B9B4 not done  23 +/− 5.8 none JEZ-2 ciA-H7/B5 A1/A3 strong 0.014 +/−0.007 not done JFK-21 ciA-F12/D12 A4/A5 not done 0.097 +/− 0.038 notdone JGA-3 ciB-A11/B5 B2/B3 not done 5.3 +/− 1.5 not done

It was observed that administration of a mixture of ciA-H7, ciA-A5, andciA-D12 in which two VHH monomers (ciA-A5 and ciA-D12) in the mixture ofmonomers were weak toxin neutralizers, resulted in subjects survivingmuch less after exposure to an amount of BoNT/A 1,000-fold BoNT/A LD₅₀(FIG. 16 panel B bottom left graph).

Thus, administration of the mixture of ciA-B5, ciA-H7, and ciA-C2 taggedmonomers, each of which are strong neutralizing VHHs, to subjectsprovided greater protection against BoNT/A than the mixture of ciA-H7,ciA-A5 and ciA-D12, in which two of the three VHH monomers do notneutralize BoNT/A. Data show that 100% of subjects administered themixture of ciA-B5, ciA-H7, and ciA-C2 with the anti-tag clearingantibody survived a dose of BoNT/A that was 1,000-fold the LD₅₀ of aBoNT/A (FIG. 16 panel B bottom left graph), and survived additional daysfollowing administration of 10,000-fold the LD₅₀ of a BoNT/A (FIG. 16panel B bottom left graph).

Complete survival (100%) was observed for subjects administered amixture of ciA-B5, ciA-H7, ciA-D12 and ciA-F12 tagged monomers andanti-tag clearing antibodies of the challenge with an amount of BoNT/Athat was 1,000-fold the LD₅₀ (FIG. 16 panel C bottom left graph).Administering a pool of anti-BoNT/A VHHs (ciA-B5, ciA-H7, ciA-D12 andciA-F12) in which only two VHHs (ciA-B5, ciA-H7) were strong toxinneutralizers only slightly delayed death in subjects exposed to1000-fold the LD₅₀ of BoNT/A (FIG. 16 panel C bottom left graph). At10,000-fold the LD₅₀ of a BoNT/A, subjects co-administered the mixtureof four VHH tagged monomers and anti-tag clearing antibody survivedadditional days compared to control subjects (FIG. 16 panel C bottomleft graph).

The antitoxin efficacy of a pool of four anti-BoNT/A VHHs taggedmonomers (ciA-A5, ciA-B5, ciA-C2 and ciA-H7) was compared to a pool ofsix different VHH tagged monomers (ciA-A5, ciA-B5, ciA-C2, ciA-H7,ciA-D12, and ciA-G5). The pool of six VHH monomers contained the sameVHHs as the pool of four VHHs and further included two VHHs (ciA-D12,and ciA-G5) that were weak neutralizers of BoNT/A (FIG. 17 and Table 5).The different pools of VHH monomers were each administered in thepresence of clearing anti-tag antibody. It was observed that 100% ofsubjects administered either the pool of four VHH tagged monomers or thepool of six VHHs tagged monomers with anti-tag clearing antibodysurvived challenge with 1000-fold the LD₅₀ of BoNT/A (FIG. 17 leftgraph). Subjects challenged with 10,000-fold the LD₅₀ of BoNT/A survivedlonger following co-administration of either the pool of four VHHmonomers or the pool of six VHH monomers with clearing anti-tagantibody, than control subjects administered only toxin (FIG. 17 rightgraph). These results show that decoration of BoNT/A with a greaternumber of VHH antibodies, four or more VHHs, greatly improved antitoxinefficacy. Administering a pool of four VHH monomers or a pool of six VHHmonomers to the subjects provided additional antitoxin efficacy comparedto administering three or fewer VHH monomers.

These data clearly show that toxin clearance was rendered much moreeffective under conditions in which BoNT is decorated by at least threeVHH antibodies and at least about three clearing antibodies. It wasobserved also that mixtures of monomers having greater number orpercentage of toxin neutralization VHHs greatly contributed to percentsurvival of subjects co-administered a vast excess of the lethal dose ofBoNT/A.

Example 16 VHH Affinity and Antitoxin Efficacy

Toxin neutralization and clearance mechanisms were observed herein todepend on affinity of antitoxin binding to the toxin. Without beinglimited by a particular theory or mechanism of action, the kinetics oftoxin binding (K_(on)) and release (K_(off)) by the antitoxin bindingagents contribute to the antitoxin efficacy.

To determine the relationship of toxin affinity to antitoxin efficacyand the role of each, assays were performed for identification ofmultiple VHHs recognizing the same epitope. In the course of anti-BoNT/AVHH screening and based on competition ELISA analysis, several VHHs(ciA-D1, ciA-H4 and ciA-H11) were identified that recognized the sameepitope as ciA-H7. SPR analysis showed that each VHH monomer recognizedand bound the ciA-H7 epitope with a different affinity. The dissociationconstant (Kd) identifies the strength of binding or affinity between aligand and a receptor, between the VHH antibody and the toxin.

The VHH Kd values for the VHHs having the stronger binding to BoNT/Awere determined to be 0.06±0.07 nM for ciA-H7, 0.71±0.004 for ciA-D1,and the VHH Kd values for the VHHs having the weakest binding to BoNT/Awere determined to be the 1.54±0.06 for ciA-H4, and 4.3±0.09 for ciA-H11respectively (FIG. 18 panel A). These four VHHs were tested withanti-tag clearing antibody for their efficacy as antitoxin VHHs incombination with the two VHHs (ciA-B5, ciA-C2) that recognize distinct,non-overlapping epitopes of BoNT/A (FIG. 18 panel B left and rightgraphs).

Subjects (five mice per group) were co-administered BoNT/A and one offour mixtures containing three VHH monomers: ciA-H7, ciA-B5 and ciA-C2;ciA-D1, ciA-B5 and ciA-C2; ciA-H4, ciA-B5 and ciA-C2; or ciA-H11, ciA-B5and ciA-C2. Each mixture included two strong neutralizing VHH monomers(ciA-B5 and ciA-C2), and one VHH of ciA-H7, ciA-D1, ciA-H4, or ciA-H11.Control subjects received toxin only.

Data show that 100% of subjects survived following co-administration of100 BoNT/A LD₅₀ and VHH mixtures containing ciA-B5 and ciA-C2 and eitherciA-H7, ciA-D1 or ciA-H4. Subjects administered the VHH mixture ofciA-B5, ciA-C2 and ciA-H11 survived the 100 LD₅₀ of BoNT/A at 80% (FIG.18 panel B left graph). Among subjects challenged with 1,000-fold theLD₅₀ of a BoNT/A (FIG. 18 panel B right graph), the level of protectionwas a function of the relative binding affinity or Kd of the VHH toBoNT/A shown in FIG. 18 panel A. Specifically the greatest protection at1,000-fold BoNT/A LD₅₀ was observed in subjects administered the VHHmixture containing ciA-B5, ciA-C2, and ciA-H7, which had the strongestBoNT/A affinity (i.e., lowest Kd value of 0.06±0.07; FIG. 18 panel A andFIG. 18 panel B right graph). The least extent of protection wasobserved in subjects administered the VHH mixture containing ciA-B5,ciA-C2, and ciA-H11 (weakest BoNT/A affinity and highest Kd value of4.3±0.09; FIG. 18 panel A and FIG. 18 panel B right graph), the survivalof which was comparable to control subjects not administered VHHmonomers.

Correlating the Kd values with antitoxin-toxin binding and affinities,it was observed that the lower the Kd value the greater the respectivetoxin affinity and the greater the antitoxin efficacy of the VHH. VHHciA-H7 was observed to have the lowest Kd and the strongest bindingaffinity to BoNT/A, and was determined to have greater antitoxinefficacy than other VHH compositions identified in FIG. 18 panel A.Thus, sub-nanomolar affinities or Kd values for the tagged toxin bindingagents is an important factor in identifying the VHH with greatestantitoxin efficacy and most effective ability to protect subjects fromtoxin-associated infection and death.

Example 17 Antitoxin VHHs Heterodimers

By engineering and expressing two anti-BoNT/A VHHs as a heterodimer, aresulting multimeric binding protein molecule was obtained, and thiscomposition was found to bind to two different sites on the toxin andyield an improved toxin affinity. Examples herein analyzed the role ofepitopic tags on the heterodimer and the role of the amount of thetagging of the heterodimer compared to the clearing antibody withrespect to increasing antitoxin efficacy of the heterodimer.

VHH heterodimers were engineered to contain an epitopic tag fordecoration of BoNT/A with two anti-tag clearing antibodies (FIG. 19panel A top drawing). Survival and protection of subjects was analyzedfollowing challenge with each of 100-fold and 1000-fold the LD₅₀ ofBoNT/A (FIG. 19 panel A bottom left and right graphs). Data show thatadministering heterodimer containing two strongly neutralizing VHHs,ciA-B5 and ciA-H7, resulted in greater antitoxin efficacy and longersurvival of subjects than administering heterodimers containing two weakor non-neutralizing VHHs, ciA-D12 and ciA-F12 (FIG. 19 panel A bottomleft and right graphs).

A second copy of the epitopic tag to the heterodimers compared to onlyone epitopic tag was observed to promote toxin decoration with fourclearing antibodies and to yield greater clearing efficacy (FIG. 19panel B top drawing). All (100%) of subjects survived a challenge witheither 1000-fold or 10,000-fold the LD₅₀ of BoNT/A and co-administrationof ciA-B5/ciA-H7 heterodimer having two epitopic tags and anti-tagclearing antibody (FIG. 19 panel B bottom graphs).

To further analyze whether two or more epitopic tags improvedheterodimer antitoxin efficacy, two sets of anti-BoNT/A VHH heterodimerswere constructed in which the two VHHs in the heterodimers were eithernon-neutralizing (ciA-D12/F12) or potent toxin neutralizing agents(ciA-B5/H7). The two different VHH heterodimers were engineeredcontaining either one or two copies of the epitopic tag (E-tag) and wereexpressed. SPR analysis confirmed that the heterodimer affinities werein the range of 10 picomolar to 100 picomolar which was significantlygreater than the affinities of the component monomers (FIG. 15 and Table5).

The antitoxin efficacies of the single tagged heterodimers administeredto mouse subjects after challenge with 1000-fold LD₅₀ of BoNT/A (FIG. 19panel A bottom left graph) were observed to be similar to resultsobtained from administering a mixture of the two corresponding monomersonly (FIG. 16 panel A bottom right graph). Administering thenon-neutralizing single-tagged heterodimer, ciA-D12/F12(1E), resulted inno protection from challenge with 1000-fold LD₅₀ of BoNT/A in theabsence of clearing antibody, and only slightly delayed death in thepresence of clearing antibody 9 FIG. 19 panel A bottom left graph). Thetoxin neutralizing single-tagged heterodimer, ciA-B5/H7(1E), delayeddeath in mice exposed to 1000 LD₅₀ BoNT/A for one to two days in theabsence of clearing antibody and efficacy was only slightly improved bythe addition of clearing antibody (FIG. 19 panel A bottom left graph).

Improved antitoxin efficacy was observed in subjects administered aheteromultimeric agent having a second copy of the epitopic tag, withboth non-neutralizing and neutralizing anti-BoNT/A VHH heterodimers inwhich the heterodimer agent was co-administered with clearing antibody.Without being limited by any particular theory or mechanism of action,it is here envisioned that component binding regions in a ‘double-taggedheterodimer’ bind at two sites on the toxin and each bound heterodimerdecorates toxin with two clearing antibodies, resulting in decoration ofthe toxin with at least four clearing antibodies (FIG. 19 panel B topdrawing) which Examples herein show had increased clearance.Administering non-neutralizing double-tagged heterodimer containingciA-D12/F12(2E) resulted in virtually no antitoxin efficacy in subjectsin the absence of clearing antibody at both 1000-fold and 10,000-foldthe LD₅₀ of BoNT/A (FIG. 19 panel B bottom left and right graphs). Inthe presence of clearing antibody, ciA-D12/F12(2E) heterodimer fullyprotected subjects (100% survival) from 100-fold BoNT/A LD₅₀ and delayeddeath about a day in subjects receiving 1000-fold BoNT/A LD₅₀ comparedto control subjects (FIG. 19 panel B bottom right graph and FIG. 20 leftgraph). Thus the presence of a second epitopic tag attached to theheterodimer dramatically improved the antitoxin efficacy.

Non-neutralizing heterodimer, ciA-D12/F12, with either one, two or threeepitopic tags was analyzed for antitoxin efficacy in the presence ofclearing antibody (FIG. 20). The single-tagged heterodimer only slightlyprotected subjects from toxin challenge of 100-fold the LD₅₀ of BoNT/A.Subjects challenged with double-tagged heterodimers and triple-taggedheterodimers were fully protected from a challenge of 100-fold the LD₅₀of BoNT/A (FIG. 20 left graph). Only little improvement in antitoxinefficacy was observed with the triple-tagged heterodimers compared tothe double-tagged heterodimers, consistent with the observation thatnear maximal clearance was achieved by decorating the target with fourclearing antibodies. A titration of the clearing antibody administeredwith the double-tagged ciA-D12/F12 heterodimer demonstrated that maximalantitoxin efficacy against both 100-fold and 1,000-fold the LD₅₀ ofBoNT/A was achieved with the number of clearing antibody molecules(measured in picomoles) administered in an amount approximatelyequivalent to the number of epitopic tags (FIG. 21 left and rightgraphs).

An even more dramatic antitoxin effect was observed in cell cultureintoxication assays using the double-tagged heterodimer, ciA-B5/H7(2E),in which both of the component anti-BoNT/A VHHs individually possesspotent neutralizing activity (FIG. 15). In the absence of clearingantibody, the double-tagged ciA-B5/H7(2E) heterodimer produced the sameantitoxin efficacy as the equivalent single-tagged heterodimer (compareFIG. 19 panel A bottom left and right graphs to FIG. 19 panel B bottomleft and right graphs). In the presence of clearing antibody, theneutralizing double-tagged heterodimer at 40 picomoles (pmoles) wasobserved to be a highly potent antitoxin that fully protected cells fromlethality when co-administered with 10,000-fold the LD₅₀ of BoNT/A,i.e., the total amount was about 3 pmoles.

A dose-response assay was performed in mouse subjects with double-taggedciA-B5/H7(2E) heterodimer co-administered with 1000-fold the LD₅₀ ofBoNT/A (FIG. 22). It was observed that both 40 pmoles and 13 pmoles ofdouble-tagged ciA-B5/H7(2E) heterodimer completely protected thesubjects against an exposure of 1000-fold the LD₅₀ of BoNT/A. A dose of4 pmoles ciA-B5/H7(2E) heterodimer had the same protective efficacy for1,000-fold the LD₅₀ of BoNT/A as a dose of 40 pmoles did with10,000-fold the LD₅₀ of BoNT/A (FIG. 15 panel B and FIG. 22). These datashow that co-administering about a fifteen-fold molar excess of thedouble-tagged heterodimer binding agent with the clearing antibody wassufficient to effectively neutralize and/or clear substantially all(greater than 99.99%) of the BoNT/A.

Example 18 Recombinant Antitoxin Efficacy in a Clinically RelevantPost-Intoxication Assay

Assays in which varying doses of toxins are co-administered withantitoxin agents were observed to permit sensitive quantification ofantitoxin efficacy. To more accurately reflect the typical clinicalsituation, antitoxin agents were tested in an assay of greater clinicalrelevance by intraperitoneally administering to mouse subjects ten-foldthe LD₅₀ of BoNT/A, and at 1.5 hours and three hours afterwards,administering intravenously neutralizing heterodimer antitoxin agentswith and without the anti-tag clearing antibody. Different sets ofanti-BoNT/A VHH heterodimers were tested: a heterodimer containingnon-neutralizing double-tagged ciA-D12/F12(2E), and a heterodimercontaining neutralizing double-tagged ciA-H7/B5(2E) heterodimer (FIG. 23panels A-B). A potent sheep anti-BoNT/A serum was used as a control inthe assay at a dose demonstrated to protect 100% of mice from lethalitygiven 10,000-fold the LD₅₀ of BoNT/A.

The non-neutralizing ciA-D12/F12(2E) heterodimer was observed to havelittle or no antitoxin efficacy in absence of clearing antibodyfollowing administration either 1.5 hours or three hours after BoNT/Achallenge (FIG. 23 panel A left and right graphs). However,ciA-D12/F12(2E) heterodimer administered with clearing antibodydisplayed an efficacy nearly equivalent to the positive control sheepantiserum (FIG. 23 panel B left and right graphs). These results showthat toxin clearance alone is sufficient to protect mice from a low doseBoNT challenge, even when administered several hours post-exposure totoxin.

Surprisingly the neutralizing ciA-H7/B5(2E) heterodimer was observed tobe as highly effective as an antitoxin in this assay, in the presence oreven absence of clearing antibody (FIG. 23 panel B). The double-taggedtoxin neutralizing heterodimer administered 1.5 hours after toxinchallenge with ten-fold the LD₅₀ of BoNT/A resulted in an antitoxinefficacy equivalent to the sheep serum polyclonal antitoxin. It wasobserved that following challenge at 10 BoNT/A LD₅₀ for 1.5 hours,subjects administered ciA-H7/B5(2E) heterodimer absent anti-tag clearingfully survived (100% survival; FIG. 23 panel B left graph). The survivalfor subjects administered ciA-H7/B5(2E) heterodimer was comparable tosubjects administered sheep antitoxin (FIG. 23 panel B left graph).

Data show that three hours after toxin challenge at ten-fold the LD₅₀ ofBoNT/A, the neutralizing ciA-H7/B5(2E) heterodimer resulted in greatersubject survival (80%) than the sheep serum polyclonal antitoxin (60%survival; FIG. 23 panel B right graph). Most important, the survival ofsubjects using neutralizing ciA-H7/B5(2E) heterodimer was the same withor without clearing antibody (FIG. 23 panel B right graph).

These data clearly show that BoNT neutralization was sufficient for fullantitoxin efficacy in a clinically relevant post-intoxication(post-exposure to toxin) assay with low dose toxin challenge. A singlerecombinant multimeric binding protein with potent toxin neutralizationproperties was as effective as antitoxin sera in a model of a typicalclinical situation involving toxin exposure and subsequent treatment.

Example 19 Antitoxin Efficacy of a Double-Tagged Heterodimer TargetingBotulinum Toxin, BoNT/B

Double-tagged VHH heterodimer antitoxins that specifically recognizedand bound unique epitopes on BoNT/B holotoxin (FIG. 13 panel B) wereidentified and expressed. Two of the VHHs, ciB-A11 and ciA-B5, wereobserved to be the most effective antitoxins of those obtained frommonomer pool assays, and were engineered and expressed as double-taggedheterodimer, ciB-A11/B5(2E).

Subjects were exposed to either 1,000-fold (FIG. 24 panel A left graph)or 10,000-fold (FIG. 24 panel A right graph) BoNT/B LD₅₀, and wereadministered a ciB-A11 and ciB-B5 heterodimer with (+αE) or without(−αE) anti-tag clearing antibody. Control subject were exposed only totoxin (no heterodimer binding proteins). Data show that in the presenceof clearing antibody the ciB-A11/B5(2E) heterodimer fully protectedsubjects challenged with 1000-fold the LD₅₀ of BoNT/B (FIG. 24 panel Aleft graph) and extended the life of subjects challenged with10,000-fold the LD₅₀ of BoNT/B (FIG. 24 panel A right graph).

Analysis was performed to determine whether the ciB-A11 and ciA-B5double tagged heterodimer was effective to treat subjects in a BoNT/Bpost-exposure in vivo model.

Subjects were intravenously exposed to 10 LD₅₀ of BoNT/A, and thenadministered 1.5 hours or three hours afterward either: ciB-A11 andciA-B5 double tagged heterodimeric protein with or without clearingantibody, or a sheep antitoxin serum. Control subjects were only exposedto 10 LD₅₀ of BoNT/B (no heterodimeric binding protein wasadministered). See FIG. 24 panel B left and right graphs. Data show 60%of subjects administered ciB-A11/B5 double tagged heterodimer withanti-tag antibody survived 1.5 hours and three hours after BoNT/Bexposure, and further that 20% more subjects survived with ciB-A11/B5double tagged heterodimer with clearing antibody treatment than withsheep antitoxin at both time points (FIG. 24 panel B left and rightgraphs). It was observed that three hours after BoNT/B exposure subjectsadministered A11/B5 double tagged heterodimer binding protein only(without anti-tag antibody) survived as long as subjects administeredsheep antitoxin (FIG. 24 panel B right graph).

Results from these clinically relevant post-intoxication assays hereinshowed that ciB-A11/B5 heterodimer with or without clearing antibody wasas effective as sheep anti-BoNT/B serum in protecting subjects fromdeath caused by BoNT/B holotoxin exposure.

Example 20 VHH Monomers Protect CT26 Cells from TcdA

Cells of murine colorectal cancer cell line CT26 were exposed to TcdA (2ng/ml) for 24 hours and to a VHH monomer specific to TcdA (A3H, SEQ IDNO 61; A11G, SEQ ID NO:63; AC1, SEQ ID NO: 62; AE1, SEQ ID NO: 64; AH3,SEQ ID NO: A1; or AA6, SEQ ID NO: 60). Controls cells were exposed toTcdA (no VHH monomer was administered). The percentage of cell roundingwas monitored using a phase contrast microscope. Control cellsadministered only TcdA showed extensive cell rounding and distorted cellmorphology associated with TcdA toxin exposure.

It was observed that each of the VHH monomers reduced the percentage ofaffected cells and protected the cells from TcdA exposure (FIG. 25). Inorder of greatest VHH monomer activity to the weakest VHH monomeractivity, the greatest activity was observed for AA6, followed AH3, AC1,A3H, AE1, and A116 respectively. It was observed that VHH monomers AA6and AH3 neutralized TcdA and protected 50% of cells from toxincytotoxicity at VHH concentrations less than about 10 nM, and thus wereconsidered to have strong TcdA neutralizing activity.

Example 21 Multimeric Binding Proteins Protect Cells from TcdA

CT26 cells were contacted to TcdA (2 ng/ml) and concentrations (0.1 nM,0.48 nM, 2.4 nM, 12 nM, 60 nM, or 300 nM) of each of VHH monomers: A3H,A11G, AC1, AE1, AH3, or AA6. Control cells were administered toxin only.The strength of each neutralizing VHH activity was observed by analyzingprotection of cells from the toxin by VHH monomers. Percentage of cellrounding (% cell affected) caused by TcdA was monitored using a phasecontrast microscope (FIG. 25). Thus, the strongest VHH produced thegreatest protection at the lowest concentration. The VHHs wereidentified in the following order of efficacy: AA6 as the strongesttherapeutic agent, followed by AH3, AC1, AE1, A11G, and then A3H asweakest therapeutic agent.

To determine whether VHH monomers or VHH multimers effectivelyneutralized TcdA, CT26 cells were exposed for 24 hours to TcdA (2 ng/ml)and different concentrations (0.03 ng/mL, 0.1 ng/mL, 1 ng/mL, 3 ng/mL,10 ng/mL, 30 ng/mL, 100 ng/mL, 300 ng/mL, or 1000 ng/mL) of VHH monomers(AH3 or AA6), VHH heterodimer containing AH3 and AA6, or a homodimer ofthe heterodimer containing the heterodimer of AH3 and AA6 and fused toan artificial homodimerization domain called oAgB (Ah3/AA6/oAgB; SEQ IDNO: 95). The oAgB domain encodes a peptide having amino acid sequenceTSPSTVRLESRVRELEDRLEELRDELERAERRANEMSIQLDEC (SEQ ID NO: 94) that bindsto proteins having the same sequence to form homodimers. The cysteine(amino acid abbreviation Cys or C) at the carboxyl end of AgBc becomesoxidized forming a covalent disulfide linkage between the two proteinmolecules to stabilize the homodimer (dimerizing sequence). Thus theAH3/AA6 heterodimer itself becomes a homodimer containing two copies ofAH3/AA6 joined by the oAgBc dimerization domain (SEQ ID NO: 95). Controlcells were exposed to toxin only and not to VHH agents. The percentageof cell rounding (% cell affected) was monitored using a phase contrastmicroscope (FIG. 26).

Data show that control cells contacted with toxin only showed extensivetoxin mediated-cell rounding, and that each of the VHH monomers, AH3/AA6heterodimer and AH3/AA6/oAgB heterodimer/homodimer neutralized TcdA andprotected the CT26 cells from the toxin (FIG. 26). The AH3/AA6/oAgBheterodimer/homodimer displayed greatest activity to neutralize andprotect cells compared to the AH3/AA6 heterodimer, AH3 monomer, and AA6monomer respectively. The AH3/AA6/oAgB heterodimer/homodimer displayedabout three-fold stronger neutralizing activity for TcdA and protectionof the cells than the AH3/AA6 heterodimer alone, and about ten-foldbetter activity and protection than the VHH monomers (AH3 and AA6respectively).

Example 22 Heterodimer Binding Proteins Protect Cells from TcdA and TcdB

To determine activity of VHH heterodimers to neutralize both TcdA andTcdB, CT26 cells were exposed overnight to TcdA (2 ng/ml) or TcdB (0.1ng/ml), and then treated with a heterodimer composition containing VHH5D and VHH AA6 (FIG. 27 left graph) or with a heterodimer compositioncontaining VHH 5D and VHH AH3 (FIG. 27 right graph). Each heterodimerwas engineered to contain a VHH(5D) that strongly neutralized TcdB (FIG.13) and to contain also a VHH (AA6 or AH3) that strongly neutralizedTcdA (FIG. 25). The percentage of cell rounding (% cell affected) wasmonitored using a phase contrast microscope (FIG. 27 left and rightgraphs).

Data show that each of the 5D/AA6 heterodimer and the 5D/AH3 heterodimerneutralized both TcdA and TcdB (FIG. 27 left and right graphs). It wasobserved that 5D/AA6 heterodimer was about five-fold more effective inneutralizing TcdA than the 5D/AH3 heterodimer. Thus, the relativeneutralization strength of each heterodimer (FIG. 27) corresponded tothe relative neutralization strength of each corresponding AA6 monomerand AH3 monomer shown in FIGS. 25-26.

It was observed that the 5D/AA6 heterodimer was about three-fold orfour-fold more effective to neutralize TcdB than the 5D/AH3 heterodimer.Using a concentration of about 0.2 nM of administered 5D/AA6heterodimer, 50% of cells were protected, compared to about 1 nM of5D/AH3 heterodimer required for this same level of protection. Withoutbeing limited by any particular theory or mechanism of action, it ishere envisioned that the relative greater TcdA neutralization ability ofthe AA6 binding region compared to AH3 binding region resulted in asynergistically greater ability of the respective heterodimer toneutralize a separate toxin TcdB. The increased toxin neutralization for5D/AA6 for TcdB is presumably caused by amino acid sequences in TcdA andTcdB that are similar and are neutralized effectively by the AA6component of the heterodimer compared to the AH3 component of theheterodimer.

Example 23 5D/AA6 Heterodimer Protected Subjects from C. difficileInfection

To further determine whether a single heterodimer could neutralize bothTcdA and TcdB and protect mice from oral C. difficile spore challenge, aprotocol for a clinically relevant mouse C. difficile infection model(Chen et al. 2008 Gastroenterology 135: 1984-1992) was performed asshown in FIG. 28. Groups of mice (ten mice/group) were treated to obtaina model of C. difficile associated diarrhea by treatment for three dayswith antibiotics in drinking water of the subjects, and then two dayslater by intraperitoneal administration of a single dose clindamycinbefore challenge with spores of a C. difficile strain expressing bothTcdA and TcdB (10⁶ spores/subject) on day zero (FIG. 28 panel A). Toinduce more severe and fulminant disease, steroid dexamethasone wassupplied to the subjects in drinking water on day −6 (100 mg/mL) untilday zero (Sun et al. 2001 Infection and Immunity 79: 2556-2864).Subjects were intraperitoneally injected with VHH heterodimer containing5D and AA6 (1 mg/kg) three times (six hours, 16 hours, and 24 hoursfollowing inoculation/challenge). Control subjects were similarlytreated by injection with PBS instead of the VHH heterodimer. Subjectswere monitored hours and days following the VHH injection.

Data show that 100% of control subjects administered toxin died withintwo days of toxin challenge (FIG. 28 panel B) and suffered diarrhea(FIG. 28 panel C). Only 20% of subjects administered 5D/AA6 heterodimerdeveloped diarrhea and 90% survived (FIG. 28 panels B and C). Thus,5D/AA6 heterodimer protected subjects from both TcdA and TcdB sporechallenge in a clinically relevant mouse C. difficile infection model.

Example 24 Recombinant Multimeric Binding Proteins Neutralize aPlurality of Disease Agents

Effectiveness of the antitoxin treatment using multimeric bindingproteins are analyzed by determine ability of the binding proteins tobind to and neutralize a disease agent target.

Recombinant heteromultimeric neutralizing binding protein containingmultiple binding regions with or without epitopic tags are produced. Thebinding regions are not identical and each binding region has affinityto specifically bind a non-overlapping portion of a disease agent: TcdAtoxin, TcdB toxin, and a Shiga toxin. The genes encoding proteins aremultimerized to form different heteromultimeric binding proteins usingthe oAgBc dimerization domain (SEQ ID NO: 94) shown in Example 21.

Subjects are exposed to a mixture of disease agents (TcdA toxin, TcdBtoxin, Shiga toxin and a norovirus), and then are administered each ofthe heteromultimeric binding proteins, or a mixture of monoclonalantibodies specific for either TcdA, TcdB, Shiga Toxin 1, and thenorovirus. Control subjects are administered the mixture of diseaseagents only (no multimeric binding proteins). Subjects are monitored forindicia of exposure to the pathogenic molecules such as diarrhea, fever,tachycardia, respiratory distress, and death.

Meyer-Kaplan plots quantifying survival of subjects are prepared andweeks later remaining subjects are sacrificed to analyze tissue and cellmorphology. A surprising synergistic protective effect is observed forsubjects administered the multimeric binding proteins with or withoutepitopic tags. Data show that subjects administered the multimericbinding proteins survive longer and have little or no indicia ofexposure to the mixture of disease agents compared results for subjectsadministered monoclonal antibodies to each disease agent and for controlsubjects administered only disease agents. Subject administeredheteromultimeric binding proteins specific for disease agents do notexperience diarrhea, fever or other indicia of exposure to the diseaseagents. Tissues from subjects administered multimeric binding proteinsshow normal cell appearance without signs of cell rounding or cell lysiscaused by either TcdA, TcdB, Shiga Toxin 1, and the norovirus. Themultimeric binding proteins neutralize each of these disease agents.Control subjects have diarrhea, and tissues excised from the intestinalsystems show indicia of colitis and extensive internal bleeding.

The multimeric binding protein specific for a mixture of bacterialtoxins and a viral infectious agent neutralize each of the diseaseagents and protected the cells from the subjects from cytotoxicity andcell lysis.

1. A pharmaceutical composition for treating a subject at risk forexposure to or exposed to at least one disease agent, the pharmaceuticalcomposition comprising: at least one recombinant heteromultimericneutralizing binding protein comprising two or more binding regions andat least one tag that is an epitope that is specifically bound by anantibody, wherein the binding regions are not identical and each bindingregion has affinity to specifically bind a non-overlapping portion ofthe disease agent, and the binding protein neutralizes the disease agentthereby treating the subject for exposure to the disease agent.
 2. Thepharmaceutical composition according to claim 1, wherein the bindingprotein is selected from the group of: a single-chain antibody (scFv); arecombinant camelid heavy-chain-only antibody (VHH); a sharkheavy-chain-only antibody (VNAR); a microprotein; a darpin; ananticalin; an adnectin; an aptamer; a Fv; a Fab; a Fab′; and a F(ab′)₂.3. The pharmaceutical composition according to claim 1 furthercomprising a linker that separates the binding regions, wherein thelinker comprises at least one selected from the group of: a peptide, aprotein, a sugar, and a nucleic acid.
 4. The pharmaceutical compositionof claim 1, wherein the disease agent is at least one selected from avirus, a cancer cell, a fungus, a bacterium, a parasite and a productthereof such as a pathogenic molecule, a protein, a lipopolysaccharide,and a toxin.
 5. The pharmaceutical composition according to claim 4,wherein the toxin is at least one selected from the group consisting of:an aflatoxin, a dinoflagellate toxin, a Botulinum toxin, aStaphylococcal α-hemolysin, a Staphylococcal leukocidin, an aerolysincytotoxic enterotoxin, a cholera toxin, a Bacillus cereus hemolysis II,an Helicobacter pylori vacuolating toxin, a Bacillus anthracis toxin, acholera toxin, an Escherichia coli serotype O157:H7 toxin, anEscherichia coli serotype O104:H7 toxin, a lipopolysaccharide endotoxin,a Shiga toxin, a pertussis toxin, a Clostridium perfringens iota toxin,a Clostridium spiroforme toxin, a Clostridium difficile toxin,Clostridium difficile toxin A, Clostridium difficile toxin B,Clostridium septicum a toxin, and Clostridium botulinum C2 toxin.
 6. Thepharmaceutical composition according to claim 1, wherein the at leastone disease agent comprises a plurality of non-identical disease agents,and the binding protein binds to the plurality of the disease agents,thereby neutralizing the disease agents.
 7. The pharmaceuticalcomposition according to claim 4, wherein the toxin is a C. botulinumtoxin, wherein the binding regions comprise recombinant camelidheavy-chain-only antibodies, and wherein the pharmaceutical compositioncomprises an amino acid sequence selected from the group consisting of:(SEQ ID NO: 56) LVQVGGSLRLSCVVSGSDISGIAMGWYRQAPGKRREMVADIFSGGSTDYAGSVKGRFTISRDNAKKTSYLQMNNVKPEDTGVYYCRLYGSGDYWGQ GTQVTVSSAHHSEDP;(SEQ ID NO: 57) LVHPGGSLRLSCAPSASLPSTPFNPFNNMVGWYRQAPGKQREMVASIGLRINYADSVKGRFTISRDNAKNTVDLQMDSLRPEDSATYYCHIEYTHY WGKGTLVTVSSEPKTPKPQ;and, (SEQ ID NO: 58) QVQLVESGGGLVQVGGSLRLSCVVSGSDISGIAMGWYRQAPGKRREMVADIFSGGSTDYAGSVKGRFTISRDNAKKTSYLQMNNVKPEDTGVYYCRLYGSGDYWGQGTQVTVSSAHHSEDPTSAIAGGGGSGGGGSGGGGSLQGQLQLVESGGGLVHPGGSLRLSCAPSASLPSTPFNPFNNMVGWYRQAPGKQREMVASIGLRINYADSVKGRFTISRDNAKNTVDLQMDSLRPEDSATYYCHIEYTHYWGKGTLVTVSSEPKTPKPQ.


8. The pharmaceutical composition according to claim 4, wherein thetoxin is a C. difficile toxin A, wherein the binding region comprises arecombinant camelid heavy-chain-only antibody having an amino acidsequence selected from the group consisting of: (SEQ ID NO: 59)QVQLVETGGLVQPGGSLRLSCAASGFTLDYSSIGWFRQAPGKEREGVSCISSSGDSTKYADSVKGRFTTSRDNAKNTVYLQMNSLKPDDTAVYYCAAFRATMCGVFPLSPYGKDDWGKGTLVTVSSEPKTPKPQP; (SEQ ID NO: 60)QLQLVETGGGLVQPGGSLRLSCAASGFTFSDYVMTWVRQAPGKGPEWIATINTDGSTMRDDSTKGRFTISRDNAKNTLYLQMTSLKPEDTALYYCARGRVISASAIRGAVRGPGTQVTVSSEPKTPKPQP; (SEQ ID NO: 61)QVQLVESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVSGISSVDGSTYYADSVRGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAADQSPIPIHYSRTYSGPYGMDYWGKGTLVTVSSAHHSEDP; (SEQ ID NO: 62)QLQLVESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVSGISFVDGSTYYADSVKGRFAISRGNAKNTVYLQMNSLKPEDTAVYYCAADQSSIPMHYSSTYSGPSGMDYWGKGTLVTVSSEPKTPKPQP; (SEQ ID NO: 63)QLQLVETGGGLVQAGGSLRLSCAASGRTLSNYPMGWFRQAPGKEREFVAAIRRIADGTYYADSVKGRFTISRDNAWNTLYLQMNGLKPEDTAVYFCATGPGAFPGMVVTNPSAYPYWGQGTQVTVSSEPKTPKPQP; (SEQ ID NO: 64)QLQLVESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVSGISSSDGSTYYADSVKGRFTISRDNATNTVYLQMNSLKPEDTAVYYCAADQAAIPMHYSASYSGPRGMDYWGKGTLVTVSSEPKTPKPQP; (SEQ ID NO: 87)MSDKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPCKMIAPILDEIADEYQGKLTVAKLNIDQNPGTAPKYGIRGIPTLLLFKNGEVAATKVGALSKGQLKEFLDANLAGSGSGHMHHHHHHSSGLVPRGSGMKETAAAKFERQHMDSPDLGTDDDDKAMAISDPNSQVQLVESGGGLVQPGGSLRLSCEASGFTLDYYGIGWFRQPPGKEREAVSYISASARTILYADSVKGRFTISRDNAKNAVYLQMNSLKREDTAVYYCARRRFSASSVNRWLADDYDVWGRGTQVAVSEEPKTPKPQTSAIAGGGGSGGGGSGGGGSLQAMAAASQVQLVESGGGLVQTGGSLRLSCASSGSIAGFETVTWSRQAPGKSLQWVASMTKTNNEIYSDSVKGRFIISRDNAKNTVYLQMNSLKPEDTGVYFCKGPELRGQGIQVTVSSEPKTPKPQPARR; and, (SEQ ID NO: 95)MSDKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPCKMIAPILDEIADEYQGKLTVAKLNIDQNPGTAPKYGIRGIPTLLLFKNGEVAATKVGALSKGQLKEFLDANLAGSGSGHMHHHHHHSSGLVPRGSGMKETAAAKFERQHMDSPDLGTDDDDKAMAISDPNSQVQLVETGGLVQPGGSLRLSCAASGFTLDYSSIGWFRQAPGKEREGVSCISSSGDSTKYADSVKGRFTTSRDNAKNTVYLQMNSLKPDDTAVYYCAAFRATMCGVFPLSPYGKDDWGKGTLVTVSSEPKTPKPQPTSAIAGGGGSGGGGSGGGGSLQAMAAAQLQLVETGGGLVQPGGSLRLSCAASGFTFSDYVMTWVRQAPGKGPEWIATINTDGSTMRDDSTKGRFTISRDNAKNTLYLQMTSLKPEDTALYYCARGRVISASAIRGAVRGPGTQVTVSSEPKTPKPQPARQTSPSTVRLESRVRELEDRLEELRDELERAERRANEMSIQLDEC.


9. The pharmaceutical composition according to claim 4, wherein thetoxin is a C. difficile toxin B, wherein the binding region comprises arecombinant camelid heavy-chain-only antibody having an amino acidsequence selected from the group consisting of: (SEQ ID NO: 65)QVQLVESGGGLVQPGGSLRLSCAASGFSLDYYGIGWFRQAPGKERQEVSYISASAKTKLYSDSVKGRFTISRDNAKNAVYLEMNSLKREDTAVYYCARRRFDASASNRWLAADYDYWGQGTQVTVSSEPKTPKPQ; (SEQ ID NO: 66)QVQLVESGGGLVQAGGSLRLSCVSSERNPGINAMGWYRQAPGSQRELVAIWQTGGSLNYADSVKGRFTISRDNLKNTVYLQMNSLKPEDTAVYYCYLKKWRDQYWGQGTQVTVSSEP KTPKPQ;(SEQ ID NO: 67)QVQLVESGGGLVQPGGSLRLSCEASGFTLDYYGIGWFRQPPGKEREAVSYISASARTILYADSVKGRFTISRDNAKNAVYLQMNSLKREDTAVYYCARRRFSASSVNRWLADDYDVWGRGTQVAVSSEPKTPKPQ; (SEQ ID NO: 68)QVQLVESGGGLVQTGGSLRLSCASSGSIAGFETVTWSRQAPGKSLQWVASMTKTNNEIYSDSVKGRFIISRDNAKNTVYLQMNSLKPEDTGVYFCKGPELRGQGIQVTVSSEPKTPKPQ;(SEQ ID NO: 69)QVQLVESGGGLVEAGGSLRLSCVVTGSSFSTSTMAWYRQPPGKQREWVASFTSGGAIKYTDSVKGRFTMSRDNAKKMTYLQMENLKPEDTAVYYCALHNAVSGSSWGRGTQVTVSSE PKTPKPQ;(SEQ ID NO: 70)VQLVESGGGLVQAGGSLRLSCAASGLMFGAMTMGWYRQAPGKEREMVAYITAGGTESYSESVKGRFTISRINANNMVYLQMTNLKVEDTAVYYCNAHNFWRTSRNWGQGTQVTVS SEPKTPKP;(SEQ ID NO: 71)VQLVESGGGLVQAGDSLTLSCAASESTENTFSMAWFRQAPGKEREYVAAFSRSGGTTNYADSVKGRATISTDNAKNTVYLHMNSLKPEDTAVYFCAADRPAGRAYFQSRSYNYWGQGTQVTVSSAHHSEDP; (SEQ ID NO: 72)VQLVESGGGSVQIGGSLRLSCVASGFTFSKNIMSWARQAPGKGLEWVSTISIGGAATSYADSVKGRFTISRDNANDTLYLQMNNLKPEDTAVYYCSRGPRTYINTASRGQGTQVTVSSEP KTPKP;(SEQ ID NO: 73)VQLVESGGGLVQAGGSLRLSCVGSGRNPGINAMGWYRQAPGSQRELVAVWQTGGSTNYADSVKGRFTISRDNLKNTVYLQMNSLKPEDTAVYYCYLKKWRDEYWGQGTQVTVSSAH HSEDP;(SEQ ID NO: 74)VQLVESGGGLVQAGESLRLSCVVSESIFRINTMGWYRQTPGKQREVVARITLRNSTTYADSVKGRFTISRDDAKNTLYLKMDSLKPEDTAVYYCHRYPLIFRNSPYWGQGTQVTVSSEPK TPKP;(SEQ ID NO: 75)VQLVESGGGLVQAGESLRLSCVVSESIFRINTMGWYRQTPGKQREVVARITLRNSTTYADSVKGRFTISRDDAKNTLYLKMDSLKPEDTAVYYCHRYPLIFRNSPYWGQGTQVTVSSEPK TP;(SEQ ID NO: 76); (SEQ ID NO: 87); and, SEQ ID NO: 95VQLVESGGGLVQAGGSLRLSCAAPGLTFTSYRMGWFRQAPGKEREYVAAITGAGATNYADSAKGRFTISKNNTASTVHLQMNSLKPEDTAVYYCAASNRAGGYWRASQYDYWGQGTQVTVSSAHHSEDP.


10. The pharmaceutical composition according to claim 4, wherein thetoxin is a Shiga toxin, wherein the binding region comprises arecombinant camelid heavy-chain-only antibody having an amino acidsequence selected from the group consisting of: (SEQ ID NO: 77)QVQLVETGGGLAQAGDSLRLSCVEPGRTLDMYAMGWIRQAPGEEREFVASISGVGGSPRYADSVKGRFTISKDNTKSTIWLQMNSLKPEDTAVYYCAAGGDIYYGGSPQWRGQGTRVT VSSEPKTPKPQ;(SEQ ID NO: 78)QVQLVESGGGLVQAGGSLRLSCAASGRINGDYAMGWERQAPGEEREEVAVNSWIGGSTYYTDSVKGRFTLSRDNAKNTLSLQMNSLKPEDTAVYYCAAGHYTDFPTYFKEYDYWGQGTQVTVSSEPKTPKPQ; (SEQ ID NO: 79)QVQLVETGGLVQAGGSLRLSCAASGVPFSDYTMAWFRQAPGKEREVVARITWRGGGPYYGNSGNGRFAISRDIAKSMVYLHMDSLKPEDTAVYYCAASRLRPALASMASDYDYWGQGTQVSVSSEPKTPKPQ; (SEQ ID NO: 80)QVQLVESGGGLVQPGESLRLSCVASASTFSTSLMGWVRQAPGKGLESVAEVRTTGGTFYAKSVAGRFTISRDNAKNTLYLQMNSLKAEDTGVYYCTAGAGPIATRYRGQGTQVTVSSA HHSEDP;(SEQ ID NO: 81)QVQLVESGGGLVQPGGSLKLSCAASGFTLADYVTVWFRQAPGKSREGVSCISSSRGTPNYADSVKGRATVSRNNANNTVYLQMNGLKPDDTAIYYCAAIRPARLRAYRECLSSQAEYDYWGQGTQVTVSSAHHSEDP; (SEQ ID NO: 82)QVQLVESGGGLVQPGGSLGLSCAMSGTTQDYSAVGWERQAPGKEREGVSCISRSGRRTNYADSVRGRFTISRDNAKDTVYLQMNSLKPDDTAVYYCAARKTDMSDPYYVGCNGMDYWGKGTLVTVSSAHHSEDP; (SEQ ID NO: 83)QVQLVESGGGLVQPGGSLTLSCTASGFTLNSYKIGWFRQAPGKEREGVSCINSGGNLRSVEGRFTISRDNTKNTVSLHMDSLKPEDTGVYHCAAAPALNVFSPCVLAPRYDYWGQGTQV TVSSAHHSEDP;(SEQ ID NO: 84)QVQLVESGGGLVQPGGSLRLSCAASGFTLGSYHIGWFRHPPGKEREGTSCLSSRGDYTKYAEAVKGRFTISRDNTKSTVYLQMNNLKPEDTGIYVCAAIRPVLSDSHCTLAARYNYWGQGTQVTVSSAHHSEDP; (SEQ ID NO: 85)QVQLVESGGGLVQPGGSLRLSCAALEFTLEDYAIAWFRQAPGKEREGVSCISKSGVTKYTDSVKGRFTVARDNAKSTVILQMNNLRPEDTAVYNCAAVRPVFVDSVCTLATRYTYWGEGTQVTVSSAHHSEDP; and (SEQ ID NO: 86)QVQLVETGGGLVQPGGSLKLSCAASEFTLDDYHIGWFRQAPGKEREGVSCINKRGDYINYKDSVKGRFTISRDGAKSTVFLQMNNLRPEDTAVYYCAAVNPVFPDSRCTLATRYTHWGQGTQVTVSSAHHSEDP.


11. The pharmaceutical composition according to claim 1, wherein thebinding protein comprises an amino acid sequence that is substantiallyidentical to at least one of SEQ ID NOs: 56-87 and 95, whereinsubstantially identical is having at least 50% identity, 60% identity,at least 65% identity, at least 70% identity, at least 75% identity, atleast 80% identity, at least 85% identity, at least 90% identity, or andat least 95% identity to the amino acid sequence of SEQ ID NOs: 56-87and
 95. 12. The pharmaceutical composition according to claim 1, furthercomprising a source of the binding protein selected from the group of: anucleic acid vector with a gene encoding the binding protein; a viralvector the binding protein; the binding protein; and the binding proteinexpressed directly from naked nucleic acid.
 13. A method for treating asubject at risk for exposure to or exposed to at least one diseaseagent, the method comprising: contacting the subject with at least onerecombinant heteromultimeric neutralizing binding protein comprising twoor more binding regions and at least one tag that is an epitope that isspecifically bound by an antibody, wherein the binding regions are notidentical, and wherein each binding region specifically binds anon-overlapping portion of the disease agent, wherein the bindingprotein neutralizes the disease agent thereby treating the subject forthe exposure.
 14. The method according to claim 13, wherein the bindingprotein is selected from the group of: a single-chain antibody (scFv); arecombinant camelid heavy-chain-only antibody (VHH); a sharkheavy-chain-only antibody (VNAR); a microprotein; a darpin; ananticalin; an adnectin; an aptamer; a Fv; a Fab; a Fab′; and a F(ab′)₂.15. The method according to claim 13, wherein the binding proteincomprises a linker that separates multimeric components of the bindingregions, wherein the linker comprises at least one selected from thegroup of: a peptide, a protein, a sugar, or a nucleic acid.
 16. Themethod of claim 13, wherein the disease agent is at least one selectedfrom a virus, a cancer cell, a fungus, a bacterium, a parasite and aproduct thereof such as a pathogenic molecule, a protein, alipopolysaccharide, and a toxin.
 17. The method according to claim 16,wherein the toxin is at least one selected from the group consisting of:a Staphylococcal α-hemolysin, a Staphylococcal leukocidin, an aerolysincytotoxic enterotoxin, a cholera toxin, a Bacillus cereus hemolysis IItoxin, an Helicobacter pylori vacuolating toxin, a Bacillus anthracistoxin, a cholera toxin, an Escherichia coli serotype O157:H7 toxin, anEscherichia coli serotype O104:H7 toxin, a lipopolysaccharide endotoxin,a Shiga toxin, a pertussis toxin, a Clostridium perfringens iota toxin,a Clostridium spiroforme toxin, a Botulinum neurotoxin, a Clostridiumdifficile toxin A, a Clostridium difficile toxin B, a Clostridiumsepticum a toxin, and a Clostridium botulinum C2 toxin; and thebacterium is selected from the group consisting of: B. anthracis, B.cereus, C. botulinum, C. difficile, C. perfringens, C. spiroforme, andV. cholera.
 18. The method according to claim 13 further comprisingobserving or detecting neutralization of the disease agent by thebinding protein and/or survival of the subject; or identifying areduction or remediation in at least one pathology symptom associatedwith the disease agent.
 19. The method according to claim 13, whereinthe disease agent comprises a plurality of disease agents, and themethod comprises prior to contacting, engineering the binding protein tobind to different domains of the plurality of the disease agents. 20.The method according to claim 13 further comprising, prior tocontacting, engineering the binding protein with at least one amino acidsequence selected from the group of SEQ ID NOs: 56-87 and
 95. 21. Themethod according to claim 13, wherein contacting the subject with thebinding protein comprises administering to the subject a source ofexpression of the binding protein, wherein the source of expression ofthe binding protein is a nucleic acid encoding the binding protein,wherein the nucleic acid comprises at least one selected from the groupconsisting of: a naked nucleic acid vector, bacterial vector, and aviral vector.
 22. A kit for treating a subject exposed to or at risk forexposure to a disease agent comprising: a pharmaceutical composition fortreating a subject at risk for exposure to or exposed to at least onedisease agent, the pharmaceutical composition comprising: at least onerecombinant heteromultimeric neutralizing binding protein comprising twoor more binding regions and at least one tag that is an epitope that isspecifically bound by an antibody, wherein the binding regions are notidentical and each binding region has affinity to specifically bind anon-overlapping portion of the disease agent, and the binding proteinneutralizes the disease agent thereby treating the subject for exposureto the disease agent; a container; and, instructions for use.
 23. Thekit according to claim 22, wherein the binding protein is selected fromthe group of: a single-chain antibody (scFv); a recombinant camelidheavy-chain-only antibody (VHH); a shark heavy-chain-only antibody(VNAR); a microprotein; a darpin; an anticalin; an adnectin; an aptamer;a Fv; a Fab; a Fab′; and a F(ab′)₂.
 24. The kit according to claim 22,wherein the binding protein comprises a tag or a linker, wherein thelinker comprises at least one selected from the group of: a peptide, aprotein, a sugar, and a nucleic acid.
 25. The kit according to claim 22,wherein the disease agent is a virus, a cancer cell, a fungus, abacterium, a parasite and a product thereof such as a pathogenicmolecule, a protein, a lipopolysaccharide, and a toxin.
 26. The kitaccording to claim 25, wherein the toxin is at least one selected fromthe group of: an aflatoxin, a dinoflagellate toxin, a Botulinum toxin, aStaphylococcal α-hemolysin, a Staphylococcal leukocidin, an aerolysincytotoxic enterotoxin, a cholera toxin, a Bacillus cereus hemolysis II,an Helicobacter pylori vacuolating toxin, a Bacillus anthracis toxin, acholera toxin, an Escherichia coli serotype O157:H7 toxin, anEscherichia coli serotype O104:H7 toxin, a lipopolysaccharide endotoxin,a Shiga toxin, a pertussis toxin, a Clostridium perfringens iota toxin,a Clostridium spiroforme toxin, a Clostridium difficile toxin,Clostridium difficile toxin A, Clostridium difficile toxin B,Clostridium septicum a toxin, and Clostridium botulinum C2 toxin. 27.The kit according to claim 25, wherein the toxin is a C. botulinumtoxin, wherein the binding regions comprise recombinant camelidheavy-chain-only antibodies, and wherein the pharmaceutical compositioncomprises an amino acid sequence selected from the group consisting of:SEQ ID NO: 56, SEQ ID NO: 57, and SEQ ID NO:
 58. 28. The kit accordingto claim 25, wherein the toxin is a C. difficile toxin A, wherein thebinding region comprises a recombinant camelid heavy-chain-only antibodyhaving an amino acid sequence selected from the group consisting of: SEQID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63,SEQ ID NO: 64, SEQ ID NO: 87, and SEQ ID NO:
 95. 29. The kit accordingto claim 25, wherein the toxin is a C. difficile toxin B, wherein thebinding region comprises a recombinant camelid heavy-chain-only antibodyhaving an amino acid sequence selected from the group consisting of: SEQID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69,SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO:74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 87, and SEQ ID NO:
 95. 30.The kit according to claim 25, wherein the toxin is a Shiga toxin,wherein the binding region comprises a recombinant camelidheavy-chain-only antibody having an amino acid sequence selected fromthe group consisting of: SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79,SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO:84, SEQ ID NO: 85, and SEQ ID NO:
 86. 31. A composition comprising atleast one amino acid sequence selected from the group of: SEQ ID NO: 56,SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO:61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ IDNO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75,SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO:80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ IDNO: 85, SEQ ID NO: 86; SEQ ID NO: 87, SEQ ID NO: 95, and substantiallyidentical, wherein substantially identical is having at least 50%identity, at least 60% identity, at least 65% identity, at least 70%identity, at least 75% identity, at least 80% identity, at least 85%identity, at least 90% identity, at least 95% identity, or at least 99%identity to the amino acid sequence of SEQ ID NOs: 56-87 and 95.